Regulation of gene expression by protein methylation

ABSTRACT

The invention relates to the cDNA and deduced amino acid sequence of the Coactivator Associated arginine (R) Methyltransferase protein, CARM1. A method is described for the use CARM1 to regulate gene expression in vivo. CARM1 has also been used to methylate arginine residues of histones, synthetic peptides, and other proteins. A method to use CARM1 to screen for drugs that inhibit its methyltransferase activity is also described, as is a method to screen for drugs that modulate CARM1&#39;s interactions with other proteins.

RELATED APPLICATION DATA

This application claims priority to provisional application Ser. No.60/112,523 filed Dec. 15, 1998, the entire disclosure of which is hereinincorporated by reference.

GOVERNMENT SUPPORT

The government may have certain rights in this invention pursuant togrants DK43093 and NS17269 from the National Institutes of Health.

FIELD OF THE INVENTION

The invention relates to coactivators of transcription and to proteinswith protein methyltransferase activity.

BACKGROUND

The activities of all cells are conducted primarily by the thousands ofdifferent types of proteins each cell produces. The blueprint or codefor synthesizing each protein is found in a corresponding gene, i.e.,each gene encodes the information needed to synthesize a specificprotein. Gene “expression” results in the production of the protein by astepwise mechanism that includes 1) “transcription” of the gene by RNApolymerase to produce a messenger RNA (mRNA) that contains the sameprotein-encoding information; and 2) “translation” of the mRNA byribosomes to produce the protein. Each gene is expressed in specifictissues and at specific times during the life of the organism.Expression of most genes is regulated in response to a variety ofsignals that arise either outside or inside the organism. This patternof specific expression for each gene is determined by the “promoterregion” of each gene, which is located adjacent to the protein-encodingregion of the gene. Each gene's promoter contains many “regulatoryelements.” Each regulatory element serves as a binding site for aspecific protein, and the binding of the appropriate protein to aspecific regulatory element can cause enhancement or repression of geneexpression. Together, the regulatory elements and the proteins that bindto these elements determine the expression pattern for the specificgene.

Hormones represent one of the most important mechanisms forcommunication between different organs and tissues in multicellularorganisms. In mammals, hormones are synthesized in one organ or tissue,and travel through the blood stream to various target organs. Byinteracting with specific receptor proteins in the target cells, thehormones change the activities of the cell. Frequently the cellulareffects of the hormone include changes in the expression of specificgenes. The protein products of these genes then carry out the biologicalactions that result in altered cellular functions.

The effects of one extremely important class of hormones are carried outby a family of related receptor proteins called the nuclear receptors(Evans, R. M. (1988) Science 240:889-895; Tsai, M-J. and B. W. O'Malley(1994) Annu. Rev. Biochem. 63:451-486; Beato, M., et al. (1995) Cell83:851-857). This family of proteins includes the receptors for all ofthe steroid hormones, thyroid hormones, vitamin D, and vitamin A, amongothers. The family also includes a large number of proteins called“orphan receptors” because they do not bind any hormone or because thehormone that binds to them is unknown; but they are neverthelessstructurally and functionally related to the hormone-binding nuclearreceptors. Nuclear receptors are transcriptional regulatory proteinsthat act by a common mechanism. For those nuclear receptors that do bindhormones, the appropriate hormone must enter the cell and bind to thenuclear receptors, which are located inside the target cells. Theactivated nuclear receptors bind to specific regulatory elementsassociated with specific genes that are regulated by these proteins.Binding of the activated nuclear receptors to the regulatory elementshelps to recruit RNA polymerase to the promoter of the gene and therebyactivates expression of the gene. This mechanism also applies to many ofthe orphan nuclear receptors.

After nuclear receptors bind to a specific regulatory element in thepromoter of the gene, they recruit RNA polymerase to the promoter by amechanism which involves another group of proteins called coactivators,that are recruited to the promoter by the nuclear receptors (Horwitz, K.B. et al. (1996) Mol. Endrocrinol. 10:1167-1177; Glass, C. K. et al.(1997) Curr. Opin. Cell Biol. 9:222-232). The complex of coactivatorshelps the receptors to activate gene expression by two differentmechanisms: 1) they make the gene more accessible to RNA polymerase byunfolding the “chromatin.” Chromatin is composed of the DNA (whichcontains all the genes) and a large group of DNA-packaging proteins. Tounfold chromatin some of the coactivator proteins contain an enzymaticactivity known as a histone acetyltransferase (HAT). HAT proteinstransfer an acetyl group from acetyl CoA to the major chromatinproteins, which are called “histones.” Acetylation of the histones helpsto unfold chromatin, thus making the gene and its promoter moreaccessible to RNA polymerase. 2) The coactivators and the nuclearreceptors make direct contact with a complex of proteins called basaltranscription factors that are associated with RNA polymerase; thisinteraction recruits RNA polymerase to the promoter. Once RNA polymerasebinds to the promoter, it initiates transcription, i.e., synthesis ofmRNA molecules. The final activation of RNA polymerase after it binds tothe promoter may also require some intervention by the coactivatorproteins, but little is known about the mechanism of these final stepsof transcriptional activation.

One specific family of three related coactivator proteins, the “nuclearreceptor coactivators” or “p160 coactivators” (because their mass isapproximately 160 kilodaltons), are required for the gene activationactivities of many of the nuclear receptor proteins. The three relatednuclear receptor coactivators are GRIP1, SRC-1, and p/CIP; all threeproteins also have additional names that are used by some investigators(Onate, S. A. et al. (1995) Science 270:1356-1357; Hong, H. et al.(1996) Proc. Natl. Acad. Sci. USA 93:4948-4952; Voegel, J. J. et al.(1996) EMBO J. 15:3667-3675; Kamei, Y. et al. (1996) Cell 85:403-414;Torchia, J. et al. (1997) Nature 387:677-684; Hong, H. et al. (1997)Mol. Cell. Biol. 17:2735-2744; Chen, H. et al. (1997) Cell 90:569-580;Anzick, S. L. et al. (1997) Science 277:965-968; Li, H. et al. (1997)Proc. Natl. Acad. Sci. USA 94:8479-8484; Takeshita, A. et al. (1997) J.Biol. Chem. 272:27629-27634). These coactivators are recruited directlyby the DNA-bound nuclear receptors. The nuclear receptor coactivators,in turn, recruit other coactivators, including CBP (or p300) and p/CAF(Chen, H. et al. 1997). All of these coactivators have been shown toplay roles in gene activation by one or both of the two mechanismsmentioned above. Some of them have HAT activities to help unfoldchromatin structure (Chen, H. et al. 1997; Spencer, T. E. et al. (1997)Nature 389:194-198), and others have been shown to make direct contactwith proteins in the RNA polymerase complex (Chen, H. et al. 1997;Swope, D. L. et al. (1996) J. Biol. Chem. 271:28138-28145). Thus, thediscovery and characterization of these coactivators provides a betterunderstanding of the mechanism by which nuclear receptors activate genetranscription.

Histones are known to be methylated as well as acetylated (Annunziato,A. T. et al. (1995) Biochem. 34:2916; Gary J. D. and Clarke, S. (1998)Prog. Nucleic Acids Res. Mol. Biol. 61:65). However, the function ofhistone methylation is unknown. Methylation of histone H3, is a dynamicprocess during the lifetime of histone molecules, and newly methylatedH3 is preferentially associated with chromatin containing acetylated H4(Annunziato, A. T. et at. 1995); thus methylation of H3, likeacetylation of H4, is associated with active chromatin. In other studieslysine methylation of histones has been found in a variety of organisms;arginine methylation of histones, while not clearly documented inmammals, has been demonstrated in other classes of organisms (Gary andClarke 1998). In Drosophila cells heat shock treatment causes increasedarginine methylation of histone H3, which could be associated withactivation of heat shock genes or repression of the other genes(Desrosiers, R. and R. M. Tanguay (1988) J. Biol. Chem. 263:4686).

Proteins can be N-methylated on amino groups of lysines and guanidinogroups of arginines or carboxymethylated on aspartate, glutamate, or theprotein C-terminus. Recent studies have provided indirect evidencesuggesting roles for methylation in a variety of cellular processes suchas RNA processing, receptor mediated signaling, and cellulardifferentiation (Aletta, J. M. et al. (1998) Trends Biochem. Sci.:23:89; Gary and Clarke 1998). However, for the most part the specificmethyltransferases, protein substrates, and specific roles played bymethylation in these phenomena have not been identified. Two types ofarginine-specific protein methyltransferase activities have beenobserved, type I and type II. Genes for three mammalian and one yeasttype I enzymes, which produce monomethyl and asymmetric dimethylarginineresidues previously have been identified (FIG. 1). On the other hand,type II protein arginine methyltransferases produce monomethyl andsymmetric dimethylarginine residues. In vitro protein substrates forvarious protein arginine methyltransferases include histones andproteins involved in RNA metabolism such as hnRNPA1, fibrillarin, andnucleolin (Lin, W-J. et al. (1996) J. Biol. Chem. 271:15034-15044.;Gary, J. D. et al. (1996) J. Biol. Chem. 271:4585; Najbauer, J. et al.(1993) J. Biol. Chem. 268:10501-10509). The arginine residues methylatedin many of these proteins are found in glycine-rich sequences, andsynthetic peptides mimicking these sequences are good substrates for thesame methyltransferases (Najbauer, J. et al. 1993).

SUMMARY

The invention relates to a transcriptional coactivator, CoactivatorAssociated arginine (R) Methyltransferase (CARM1).

One aspect of the invention includes CARM1 cDNA polynucleotides such as(SEQ ID NO: 1). Polynucleotides include those with sequencessubstantially equivalent to SEQ ID NO: 1, including fragments thereof.Polynucleotides of the present invention also include, but are notlimited to, a polynucleotide complementary to the nucleotide sequence ofSEQ ID NO: 1.

Polynucleotides according to the invention have numerous applications ina variety of techniques known to those skilled in the art of molecularbiology. These techniques include use as hybridization probes, use asoligomers, i.e. primers for PCR, use for chromosome and gene mapping,use in the recombinant production of protein, and use in generation ofantisense DNA or RNA, their chemical analogs and the like. For example,when the expression of an MRNA is largely restricted to a particularcell or tissue type, polynucleotides of the invention can be used ashybridization probes to detect the presence of the specific MRNA in theparticular cell or tissue RNA using, e.g., in situ hybridization. Theinvention also includes vectors encoding the polynucleotides of theinvention.

The invention also describes the deduced amino acid sequence of theCARM1 protein (SEQ ID NO: 2). The invention also describes isolatedCARM1 proteins.

The polypeptides according to the invention can be used in a variety ofprocedures and methods that are currently applied to other proteins. Forexample, a polypeptide of the invention can be used to generate anantibody that specifically binds the polypeptide. The inventiondescribes antibodies that specifically interact with the CARM1 proteinor fragments thereof.

The polypeptides of the invention also act as methyltransferases ofhistones and other proteins and can therefore be used for the study ofmethylation processes in transcription and to methylate amino acidresidues within histones and other proteins.

Methylated proteins produced by the methods of the invention can be usedto identify demethylating enzymes. Methylated histones, for example, canbe used to screen for demethylating enzymes.

The methods of the present invention further relate to the methods fordetecting the presence of the polynucleotides or polypeptides of theinvention in a sample. Such methods can, for example, be utilized as aprognostic indicator of diseases that involve CARM1, modified forms ofCARM1, or altered expression of CARM1.

Methods are also provided for identifying proteins that interact withCARM1 as well as methods for screening of drugs that alter CARM1'sinteractions with other proteins.

Another aspect of the invention is to provide methods to screen formolecules that alter CARM1 methyltransferase activity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a comparison of the region of highest homology betweenCARM1, three other mammalian protein arginine methyltransferases (Lin,W-J. et al. 1996; Tang, J. et al. (1998) J. Biol. Chem. 273:16935;Scott, H. S. et al. (1998) Genomics 48:330.) and one yeast proteinarginine methyltransferase (Gary, J. D. et al. 1996); the sequences areshown, with dashes (-) representing the same amino acid as in CARM1 anddots (.) representing spaces inserted for optimum alignment. Thelocation of a VLD-to-AAA mutation used in these studies is indicated.

FIG. 2 shows the expression of CARM1 mRNA in various adult mouse tissuesas examined by hybridizing a 0.6-kb BamHI cDNA fragment (representingCARM1 codons 3-198) to a multiple tissue northern blot (Clontech) asdescribed previously (Hong, H. et al. 1997). Positions of RNA sizemarkers are shown on the left.

FIG. 3 shows the binding in vitro of CARM1 (SEQ ED NO: 2) and a CARM1variant (SEQ ED NO: 3) to the C-terminal region of p160 coactivators.

FIG. 4 shows binding of CARM1 to GRIP1 in vivo, i.e. in living yeast.Sub-fragments of the GRIP1 C-terminal domain (GRIP1_(c)), fused with theGal4DBD, were tested in the yeast two-hybrid system as describedpreviously (Ding, X. F. et al. (1998) Mol. Endocrinol. 12:302) forbinding to CARM1 or to α-actinin, fused to Gal4AD. β-galactosidase(β-gal) activity indicates interaction between the two hybrid proteins.

FIG. 5 shows the enhancement by CARM1 of reporter gene activation byGal4DBD-GRIP1_(c). A) CV-1 cells in 6-well dishes (3.3 cm diameter well)were transiently transfected with 0.5 of μg pM.GRIP1_(c) (coding forGal4DBD-GRIP1_(c) where GRIP1_(c) is GRIP1 amino acids 1121-1462), 0.5μg of GK1 reporter gene (luciferase gene controlled by Gal4 bindingsites) (Webb, P. et al., (1998) Mol. Endocrinol. 12:1605), and 0-0.8 μgof pSG5.HA-CARM1, using Superfectin (Qiagen) according to manufacturer'sprotocol. Total DNA was adjusted to 2.0 μg per well with the appropriateamount of pSG5. Cell extracts were prepared approximately 48 h aftertransfection and assayed with Promega Luciferase Assay kit. Relativelight units of luciferase activity presented are the mean and standarddeviation of three transfected wells. B) CV-1 cells were transfected asin A with the indicated amount of pM.GRIP1_(c) and zero or 0.5 μg ofpSG5.HA-CARM1.

FIG. 6 shows the enhancement by CARM1 of reporter gene activation bynuclear receptors and the elimination of CARM1 coactivator function bythe VLD-to-AAA mutation. (A) Transient transfection assays with CV-1cells were performed as in FIG. 5 with 0.5 μg of GK1 reporter gene and0.5 μg of each of the indicated vectors. (B) CV-1 cells were transientlytransfected as in FIG. 5 with the following vectors, as indicated: 0.5μg of nuclear receptor expression vector pSVAR₀ (Brinkanann, A. O. etal. (1989) J. Steroid Biochem. Molec. Biol. 34:307) expressing AR, pHE0(Green, S. et al. (1988) Nucleic Acids Res. 16:369) expressing ER, orpCMX.hTRβ1 (Feng, W. et al. (1998) Science 280:1747) expressing TR; 0.5μg of a luciferase reporter gene with an appropriate promoter, MMWVpromoter for AR, or MMTV promoter with the native glucocorticoidresponse elements replaced by a single estrogen response element for ERor palindromic thyroid hormone response element for TR (Umesono, K. andR. M. Evans (1989) Cell 57:1139); 0.5 μg of pSG5.HA-GRIP1; and 0.5 μg ofpSG5.HA-CARM1 or pSG5.HA-CARM1(VLD mutant). Transfection efficiency wasmonitored by using βgalactosidase activity expressed from 0.1 μg ofco-transfected pCMV-βgal vector (Hong, H. et al. (1996) Proc. Natl.Acad. Sci. USA 93:4948-4952) as an internal control. After transfection,cells were grown in charcoal-treated serum; where indicated 20 nMhormone (H), i.e. dihydrotestosterone for AR, estradiol for ER, ortriiodothyronine for TR, was included during the last 40 h of culture.The data is representative of three independent experiments.

FIG. 7 shows a model for primary and secondary coactivators of nuclearreceptors (NR). Nuclear receptor dimers bind directly to the hormoneresponse element (HRE) and activate transcription by recruitingcoactivators, which open chromatin structure (signified by nucleosome)and recruit a transcription initiation complex (TIC), composed of RNApolymerase II (Pol II), basal transcription factors such as TBP andTFIIB, and a large complex of accessory proteins (Chang, M. and J. A.Jaehning (1997) Nucleic Acids Res. 25:4861). GRIP1 and other p160 familymembers serve as primary coactivators in this case, binding directly tothe NRs. CBP, p/CAF, and CARM1 are recruited by the primary coactivatorsand thus serve as secondary coactivators. Some coactivators (e.g. CBP)may help to recruit the TIC through direct interactions with basaltranscription factors. Some coactivators (e.g. CBP and p/CAF) canacetylate histones, using acetyl-CoA (AcCoA). We propose that CARM1'scoactivator activity is due to its ability to methylate histones orother proteins in chromatin or the transcription initiation complex,using S-adenosylmethionine (SAM) as methyl donor.

FIG. 8 shows the histone methyltransferase activity of CARM1. CARM1 andPRMT1 have different protein methyltransferase substrate specificities.CARM1 methylates histone H3, whereas PRMT1 methylates histone H4. PRMT1was also previously shown to methylate other proteins, including hnRNPA1, but had not previously been shown to methylate histone H4. (A,B)Calf thymus histones (Boehringer Mannheim) were incubated for 30 min at30° C. in 32.5 μl reactions containing 20 mM Tris-Cl, 0.2 M NaCl, 4 mMEDTA, pH 8.0, 0.32 mg/ml individual histone (2a, 2b, 3, or 4) or 1.3mg/ml mixed histone (M), 0.037 mg/ml GST-CARM1 or GST-PRMT1, and 7 μMS-adenosyl-L-[methyl-³H]methionine (specific activity of 14.7 Ci/mmol).Reactions were stopped by addition of SDS-NuPAGE sample buffer (Novex),and 40% of each stopped reaction was then subjected to SDS-PAGE in 4-12%NuPAGE Bis-Tris gradient gels (Novex) using the Na-MES running buffer.Gels were stained with Coomassie Blue R-250 (A), and then subjected tofluorography (Chamberlin, M. (1978) Anal. Biochem. 98:132) for 12 h at−70 ° C. on sensitized Kodak XAR-5 film (B). Molecular weight markers(MW) are shown at left. Concentrations of GST fusion proteins weredetermined in comparison with bovine serum albumin standards (Sigma) bySDS polyacrylamide gel electrophoresis and Coomassie Blue staining; itwas assumed that bovine serum albumin stained twice as intensely as mostother proteins. Concentrations of histones and hnRNP Al were determinedby the method of Lowry (Lowry, O. H. et al. (1951) J. Biol. Chem193:265). (C) Methylation and electrophoresis were carried out asdescribed above except that protein substrates were 2.7 mg/ml mixedhistone (Hiw), 0.083 mg/ml hnRNP-A1 (A1), or no substrate (−), and theconcentrations of GST-CARM1, GST-CARM1 VLD mutant (VLD), and GST-PRMT1,were 0.05,0.02, and 0.03 mg/ml respectively. Two different preparationsof the GST-CARM1 VLD mutant failed to show detectable activity towardsany substrate. Recombinant human hnRNP A1 expressed in E. coli (Mayeda,A. A. R. and Krainer (1995) Cell 68:365) was kindly provided by Dr. A.Krainer (Cold Spring Harbor Laboratories, N.Y.).

FIG. 9 shows that PRMT1 can also serve as a coactivator for nuclearreceptors. Furthermore, CARM1 and PRMT1 act cooperatively as enhancersof nuclear receptor function, i.e. the two together are at least aseffective or more effective than the sum of their individual activities.Transient transfections were performed as in FIG. 5. CV-1 cells weretransfected with the following plasmids: expression vector for nuclearreceptor (0.1 μg pSVAR₀ for androgen receptor [AR], 0.001 μg ofpCMX.TRβ1 for thyroid receptor [TR], or 0.001 μg of pHE0 for estrogenreceptor [ER]), 0.25 μg of reporter gene for each nuclear receptor asdescribed in FIG. 6B, 0.25 μg of pSG5.HA-GRIP1, and the indicated amountof plasmids encoding CARM1 or PRMT1.

FIG. 10 shows that at low levels of nuclear receptor expression, thehormone dependent activity of the nuclear receptors depends almostentirely on the presence of three different coactivators, at least oneof which is a protein methyltransferase. Several different combinationsof three coactivators work: A) Orphan nuclear receptors ERR3 and ERR1,which require no ligand, are active without exogenously addedcoactivators when high levels of these nuclear receptors are expressed;but when low levels of these nuclear receptors are expressed (1 ng ofexpression plasmid in FIG. 10A), GRIP1+CARM1+PRMT1 is required foractivity (226-fold over controls for ERR3 and 13.8-fold over controlsfor ERR1). Omission of any one of these coactivators almost completelyeliminated activity. p160 coactivators other than GRIP1 could besubstituted for GRIP1 with similar results. B) When high levels ofestrogen receptor are expressed in CV-1 cells (using 100 ng of ERexpression vector), the estrogen receptor alone is active, and theactivity is enhanced by GRIP1 alone or GRIP1+one other coactivator (p300or CARM1) (right side of panel). However, when low levels of estrogenare expressed (using 1-10 ng of ER expression vector) BR alone is almostinactive, and individual coactivators or combinations of any twocoactivators cause little stimulation; activity is almost entirelydependent on the presence of GRIP1+CARM1+p300 (left side of panel).

FIG. 11 shows that PRMT2 and PRMT3 also serve as coactivators fornuclear receptors. CV-1 cells were transiently transfected withexpression vectors for the orphan (i.e. no ligand) nuclear receptorERR3, and where indicated the expression vectors for GRIP1, CARM 1,PRMT1, PRMT2, and PRMT3. Like PRMT1 (FIGS. 9 & 10), PRMT2 and PRMT3could enhance nuclear receptor function in cooperation with CARM1.

DETAILED DESCRIPTION Definitions

The term “nucleotide sequence” refers to a heteropolymer of nucleotidesor the sequence of nucleotides. One of skill in the art will readilydiscern from contextual cues which of the two definitions isappropriate. The terms “nucleic acid,” “nucleic acid molecule” and“polynucleotide” are also used interchangeably herein to refer to aheteropolymer of nucleotides. Generally, nucleic acid segments providedby this invention may be assembled from fragments of the genome andshort oligonucleotide linkers, or from a series of oligonucleotides, orfrom individual nucleotides, to provide a synthetic nucleic acid whichis capable of being expressed in a recombinant transcriptional unitcomprising regulatory elements derived from a microbial or viral operon,or a eukaryotic gene.

The terms “oligonucleotide fragment” or a “polynucleotide fragment,”“portion,” or “segment” refer to a stretch of nucleotide residues whichis long enough to use in polymerase chain reaction (PCR) or varioushybridization procedures to identify or amplify identical or relatedparts of mRNA or DNA molecules.

“Oligonucleotides” or “nucleic acid probes” are prepared based on thepolynucleotide sequences provided herein. Oligonucleotides compriseportions of such a polynucleotide sequence having at least about 15nucleotides and usually at least about 20 nucleotides. Nucleic acidprobes comprise portions of such a polynucleotide sequence having fewernucleotides than about 3 kb, usually fewer than 1 kb. After appropriatetesting to eliminate false positives, these probes may, for example, beused to determine whether specific MRNA molecules are present in a cellor tissue.

The term “probes” includes naturally occurring or recombinant orchemically synthesized single- or double-stranded nucleic acids. Theymay be labeled by nick translation, Klenow fill-in reaction, PCR orother methods well known in the art. Probes of the present invention,their preparation and/or labeling are elaborated in Sambrook, J. et al.,1989. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.;or Ausubel, F. et al., 1989, Current Protocols in Molecular Biology,John Wiley & Sons, New York, both of which are incorporated herein byreference in their entirety.

The term “recombinant,” when used herein to refer to a polypeptide orprotein, means that a polypeptide or protein is derived from recombinant(e.g., microbial, mammalian, or insect-based) expression systems.“Microbial” refers to recombinant polypeptides or proteins made inbacterial or fungal (e.g., yeast) expression systems. As a product,“recombinant microbial” defines a polypeptide or protein essentiallyfree of native endogenous substances and unaccompanied by associatednative glycosylation. Polypeptides or proteins expressed in mostbacterial cultures, e.g., E. coli, will be free of glycosylationmodifications; polypeptides or proteins expressed in yeast may have aglycosylation pattern in general different from those expressed inmammalian cells.

The term “recombinant expression vector” refers to a plasmid or phage orvirus or vector, for expressing a polypeptide from a polynucleotidesequence. An expression vector can comprise a transcriptional unitcomprising an assembly of: 1) a genetic element or elements having aregulatory role in gene expression, for example, promoters or enhancers,2) a structural or coding sequence which is transcribed into mRNA andtranslated into protein, and 3) appropriate transcription initiation andtermination sequences. It may include an N-terminal methionine residue.This residue may or may not be subsequently cleaved from the expressedrecombinant protein to provide a final product.

The term “recombinant expression system” means host cells which havestably integrated a recombinant transcriptional unit into chromosomalDNA or carry the recombinant transcriptional unit extrachromosomally.Recombinant expression systems as defined herein will expressheterologous polypeptides or proteins upon induction of the regulatoryelements linked to the DNA segment or synthetic gene to be expressed.This term also encompasses host cells which have stably integrated arecombinant genetic element or elements having a regulatory role in geneexpression, for example, promoters or enhancers. Recombinant expressionsystems as defined herein will express polypeptides or proteinsendogenous to the cell upon induction of the regulatory elements linkedto the endogenous DNA segment or gene to be expressed. The cells can beprokaryotic or eukaryotic.

The term “open reading frame,” or “ORF,” means a series of nucleotidetriplets coding for amino acids without any termination codons and is asequence translatable into protein.

The term “active” refers to those forms of the polypeptide which retaina biologic and/or immunologic activity or activities of any naturallyoccurring polypeptide. An active polypeptide can possess one activity ofa polypeptide, but not another, e.g. possess p160 binding activity butlack methyltransferase activity.

The term “naturally occurring polypeptide” refers to polypeptidesproduced by cells that have not been genetically engineered andspecifically contemplates various polypeptides arising frompost-translational modifications of the polypeptide including, but notlimited to, acetylation, carboxylation, glycosylation, phosphorylation,lipidation and acylation.

The term “derivative” refers to polypeptides chemically modified by suchtechniques as ubiquitination, labeling (e.g., with radionuclides orvarious enzymes), pegylation (derivatization with polyethylene glycol)and insertion or substitution by chemical synthesis of amino acids suchas ornithine, which do not normally occur in human proteins.

The term “recombinant variant” refers to any polypeptide differing fromnaturally occurring polypeptides by amino acid insertions, deletions,and substitutions, created using recombinant DNA techniques. Guidance indetermining which amino acid residues may be replaced, added or deletedwithout abolishing activities of interest, such as catalytic activity,may be found by comparing the sequence of the particular polypeptidewith that of homologous peptides and minimizing the number of amino acidsequence changes made in regions of high homology.

Preferably, amino acid “substitutions” are the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, i.e., conservative amino acid replacements. Aminoacid substitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methonine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamnine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutainic acid. “Insertions” or“deletions” are typically in the range of about 1 to 5 amino acids. Thevariation allowed may be experimentally determined by systematicallymaking insertions, deletions, or substitutions of amino acids in apolypeptide molecule using recombinant DNA techniques and assaying theresulting recombinant variants for activity.

Alternatively, where alteration of function is desired, insertions,deletions or non-conservative alterations can be engineered to producepolypeptide variants. Such variants can, for example, alter one or moreof the biological functions or biochemical characteristics of thepolypeptides of the invention. For example, such alterations may changepolypeptide characteristics such as ligand-binding affinities,interchain affinities, or degradation/turnover rate. Further, suchalterations can be selected so as to generate polypeptides that arebetter suited for expression, scale up and the like in the host cellschosen for expression. For example, cysteine residues can be deleted orsubstituted with another amino acid residue in order to eliminatedisulfide bridges. A variant's catalytic efficiency can be diminishedthrough deletion or non-conservative substitution of residues importantfor catalysis.

As used herein, “substantially equivalent” can refer both to nucleotideand amino acid sequences, for example a mutant sequence, that variesfrom a reference sequence by one or more substitutions, deletions, oradditions, the net effect of which does not result in an adversefunctional dissimilarity between the reference and subject sequences.Typically, such a substantially equivalent sequence varies from one ofthose listed herein by no more than about 20% (i.e., the number ofindividual residue substitutions, additions, and/or deletions in asubstantially equivalent sequence, as compared to the correspondingreference sequence, divided by the total number of residues in thesubstantially equivalent sequence is about 0.20 or less). Such asequence is said to have 80% sequence identity to the listed sequence.In one embodiment, a substantially equivalent, e.g., mutant, sequence ofthe invention varies from a listed sequence by no more than 20% (80%sequence identity); in a variation of this embodiment, by no more than10% (90% sequence identity); and in a further variation of thisembodiment, by no more than 5% (95% sequence identity). Substantiallyequivalent, e.g., mutant, amino acid sequences according to theinvention generally have at least 80% sequence identity with a listedamino acid sequence.

A polypeptide “fragment,” “portion,” or “segment” is a stretch of aminoacid residues of at least about 5 amino acids, often at least about 7amino acids, typically at least about 9 to 13 amino acids, and, invarious embodiments, at least about 17 or more amino acids. To beactive, any polypeptide must have sufficient length to display biologicand/or immunologic activity.

Alternatively, recombinant variants encoding these same or similarpolypeptides may be synthesized or selected by making use of the“redundancy” in the genetic code. Various codon substitutions, such asthe silent changes which produce various restriction sites are wellknown in the art and may be introduced to optimize cloning into aplasmid or viral vector or expression in a particular prokaryotic oreukaryotic system. Mutations in the polynucleotide sequence may bereflected in the polypeptide or domains of other peptides added to thepolypeptide to modify the properties of any part of the polypeptide, tochange characteristics such as ligand-binding affinities, interchainaffinities, or degradation/turnover rate.

The term “purified” as used herein denotes that the indicated nucleicacid or polypeptide is present in the substantial absence of otherbiological macromolecules, e.g., polynucleotides, proteins, and thelike. In one embodiment, the polynucleotide or polypeptide is purifiedsuch that it constitutes at least 95% by weight, more preferably atleast 99.8% by weight, of the indicated biological macromoleculespresent (but water, buffers, and other small molecules, especiallymolecules having a molecular weight of less than 1000 daltons, can bepresent).

The term “isolated” as used herein refers to a nucleic acid orpolypeptide separated from at least one other component (e.g., nucleicacid or polypeptide) present with the nucleic acid or polypeptide in itsnatural source. In one embodiment, the nucleic acid or polypeptide isfound in the presence of (if anything) only a solvent, buffer, ion, orother component normally present in a solution of the same. The terms“isolated” and “purified” do not encompass nucleic acids or polypeptidespresent in their natural source.

The term “infection” refers to the introduction of nucleic acids into asuitable host cell by use of a virus or viral vector. The term“transformation” means introducing DNA into a suitable host cell so thatthe DNA is replicable, either as an extrachromosomal element, or bychromosomal integration. The term “transfection” refers to the taking upof an expression vector by a suitable host cell, whether or not anycoding sequences are in fact expressed.

Each of the above terms is meant to encompasses all that is describedfor each, unless the context dictates otherwise.

POLYNUCLEOTIDES AND NUCLEIC ACIDS OF THE INVENTION

All The invention provides polynucleotides substantially equivalent toSEQ ID NO: 1, which is the CDNA encoding the polypeptide sequence, SEQID NO: 2. The present invention also provides genes corresponding to thecDNA sequences disclosed herein. The corresponding genes can be isolatedin accordance with known methods using the sequence informationdisclosed herein. Such methods include the preparation of probes orprimers from the disclosed sequence information for identificationand/or amplification of genes in appropriate genomic libraries or othersources of genomic materials.

The compositions of the present invention include isolatedpolynucleotides, including recombinant DNA molecules, cloned genes ordegenerate variants thereof, especially naturally occurring variantssuch as allelic variants, novel isolated polypeptides, and antibodiesthat specifically recognize one or more epitopes present on suchpolypeptides.

The polynucleotides of the invention also include nucleotide sequencesthat are substantially equivalent to the polynucleotides recited above.Polynucleotides according to the invention can have at least about 80%,more typically at least about 90%, and even more typically at leastabout 95%, sequence identity to a polynucleotide recited above. Theinvention also provides the complement of the polynucleotides includinga nucleotide sequence that has at least about 80%, more typically atleast about 90%, and even more typically at least about 95%, sequenceidentity to a polynucleotide encoding a polypeptide recited above. Thepolynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) orRNA such as mRNA or an antisense RNA. Methods and algorithms forobtaining such polynucleotides are well known to those of skill in theart and can include, for example, methods for determining hybridizationconditions which can routinely isolate polynucleotides of the desiredsequence identities.

A polynucleotide according to the invention can be joined to any of avariety of other nucleotide sequences by well-established recombinantDNA techniques (see Sambrook J et al. (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, NY). Useful nucleotidesequences for joining to polypeptides include an assortment of vectors,e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and thelike, that are well known in the art. Accordingly, the invention alsoprovides a vector including a polynucleotide of the invention and a hostcell containing the polynucleotide. In general, the vector contains anorigin of replication functional in at least one organism, convenientrestriction endonuclease sites, and a selectable marker for the hostcell. Vectors according to the invention include expression vectors,replication vectors, probe generation vectors, and sequencing vectors. Ahost cell according to the invention can be a prokaryotic or eukaryoticcell and can be a unicellular organism or part of a multicellularorganism.

The sequences falling within the scope of the present invention are notlimited to the specific sequences herein described, but also includeallelic variations thereof Allelic variations can be routinelydetermined by comparing the sequence provided in SEQ ID NO: 1, arepresentative fragment thereof, or a nucleotide sequence at least 98%identical to SEQ ID NO: 1, with a sequence from another murine isolate.An allelic variation is more typically at least 99% identical to SEQ IDNO: 1 and even more typically 99.8% identical to SEQ ID NO: 1.Furthermore, to accommodate codon variability, the invention includesnucleic acid molecules coding for the same amino acid sequences as dothe specific ORFs disclosed herein. In other words, in the coding regionof an ORF, substitution of one codon for another which encodes the sameamino acid is expressly contemplated. Any specific sequence disclosedherein can be readily screened for errors by resequencing a particularfragment, such as an ORF, in both directions (i.e., sequence bothstrands).

The present invention further provides recombinant constructs comprisinga nucleic acid having the sequence of SEQ ID NO: 1 or a fragmentthereof. The recombinant constructs of the present invention comprise avector, such as a plasmid or viral vector, into which a nucleic acidhaving the sequence of SEQ ID NO: 1 or a fragment thereof is inserted,in a forward or reverse orientation. In the case of a vector comprisingone of the ORFs of the present invention, the vector may furthercomprise regulatory sequences including, for example, a promoteroperably linked to the ORF. Large numbers of suitable vectors andpromoters are known to those of skill in the art and are commerciallyavailable for generating the recombinant constructs of the presentinvention.

The nucleic acid sequences of the invention are further directed tosequences which encode variants of the described nucleic acids. Theseamino acid sequence variants may be prepared by methods known in the artby introducing appropriate nucleotide changes into a native or variantpolynucleotide. There are two variables in the construction of aminoacid sequence variants: the location of the mutation and the nature ofthe mutation. The amino acid sequence variants of the nucleic acids arepreferably constructed by mutating the polynucleotide to give an aminoacid sequence that does not occur in nature. In a preferred method,polynucleotides encoding the novel nucleic acids are changed viasite-directed mutagenesis.

Use of Nucleic Acids as Probes

Another aspect of the subject invention is to provide forpolypeptide-specific nucleic acid hybridization probes capable ofhybridizing with naturally-occurring nucleotide sequences. Thehybridization probes of the subject invention may be derived from thenucleotide sequence of SEQ ID NO: 1, fragments or complements thereof.Because the corresponding gene is only expressed in a limited number oftissues, a hybridization probe derived from SEQ ID NO: 1 can be used asan indicator of the presence of RNA of cell type of such a tissue in asample as shown in Example 1.

Such probes may be of recombinant origin, may be chemically synthesized,or a mixture of both. The probe will comprise a discrete nucleotidesequence for the detection of identical sequences or a degenerate poolof possible sequences for identification of closely related genomicsequences. Other means for producing specific hybridization probes fornucleic acids include the cloning of nucleic acid sequences into vectorsfor the production of MnRNA probes.

Hosts

The present invention further provides host cells genetically engineeredto contain the polynucleotides of the invention. For example, such hostcells may contain nucleic acids of the invention introduced into thehost cell using known transformation, transfection or infection methods.The present invention still further provides host cells geneticallyengineered to express the polynucleotides of the invention, wherein suchpolynucleotides are in operative association with a regulatory sequenceheterologous to the host cell which drives expression of thepolynucleotides in the cell.

The host cell can be a higher eukaryotic host cell, such as a mammaliancell or an insect cell, a lower eukaryotic host cell, such as a yeastcell, or the host cell can be a prokaryotic cell, such as a bacterialcell. Introduction of the recombinant construct into the host cell canbe effected by calcium phosphate transfection, DEAE, dextran mediatedtransfection, or electroporation (Davis, L. et al., Basic Methods inMolecular Biology (1986)). The host cells containing one ofpolynucleotides of the invention, can be used in conventional manners toproduce the gene product encoded by the isolated fragment (in the caseof an ORF) or can be used to produce a heterologous protein under thecontrol of an appropriate promoter region.

Any host/vector system can be used to express one or more of the ORFs ofthe present invention. These include, but are not limited to, eukaryotichosts such as HeLa cells, CV-1 cell, COS cells, and Sf9 cells, as wellas prokaryotic host such as E. coli and B. subtilis. The most preferredcells are those which do not normally express the particular polypeptideor protein or which expresses the polypeptide or protein at low naturallevel. Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al., inMolecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y. (1989).

Regulation of Transcription

Polynucleotides of the invention and vectors capable of expressing thesepolynucleotides are useful for the regulation of transcription in cells.

Increased expression of CARM1 in cells enhances the function of nuclearreceptor coactivators of the p160 family including GRIP1, SRC-1, andp/CIP. CARM1 expression in mammalian cells enhances the activity of fulllength GRIP1 or of the C-terminal domain of GRIP1 attached to the DNAbinding domain of a heterologous protein. Increased expression of CARM1in cells, in conjunction with increased expression of coactivators ofthe GRIP1 family, enhances the function of nuclear receptors. Theenhancement by CARM1 is over and above that achieved by the increasedexpression of a GRIP1-type coactivator. Thus, CARM1 can serve as acoactivator for nuclear receptors.

The activity of other transcriptional activator proteins that rely onGRIP1-type coactivators will be enhanced by increased expression ofCARM1. Examples of other transcriptional activator proteins that may useGRIP1-type coactivators are other nuclear receptors, AP1, and STATs(Glass C K et al. (1997) Curr. Opin. Cell Biol. 9:222-232; Kamei Y etal. (1996) Cell 85:403-414; Korzus E et al. (1998) Science 279:703-707;Yao T-P et al. (1996) Proc. Natl. Acad. Sci. USA 93:10626-10631).

CARM1 polynucleotides or polypeptides can also be used in conjunctionwith other transcriptional activating molecules to increasetranscription of a nuclear receptor-dependent gene. In one embodiment,CARM1 is expressed simultaneously with a histone acetyl transferase(HAT). Transcription of a gene under the control of a nuclear receptoris synergistically enhanced by the presence of CARM1 and a HAT.

Gene Therapy

Polynucleotides of the present invention can also be used for genetherapy for the treatment of disorders which are mediated by CARM1,certain hormones, such as those that act as ligands for nuclear hormonereceptors, or by nuclear hormone receptors. Such therapy achieves itstherapeutic effect by introduction of the appropriate CARM1polynucleotide (e.g., SEQ ID NO: 1) which contains a CARM1 gene (senseor antisense), into cells of subjects having the disorder to increase ordecrease CARM1 activity in the subjects' cells. Delivery of sense orantisense CARM1 polynucleotide constructs can be achieved using arecombinant expression vector such as a chimeric virus or a colloidaldispersion system. An expression vector including the CARM1polynucleotide sequence may be introduced to the subject's cells ex vivoafter removing, for example, stem cells from a subject's bone marrow.The cells are then reintroduced into the subject, (e.g., into subject'sbone marrow).

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuIV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), Rous SarcomaVirus (RSV), and gibbon ape leukemia virus (GaLV), which provides abroader host range than many of the murine viruses. A number ofadditional retroviral vectors can incorporate multiple genes. All ofthese vectors can transfer or incorporate a gene for a selectable markerso that transduced cells can be identified and selected for. Byinserting a CARM1 sequence of interest into the viral vector, along withanother gene which encodes the ligand for a receptor on a specifictarget cell, for example, the vector is now target specific. Preferredtargeting is accomplished by using an antibody to target the retroviralvector. Those of skill in the art will know of, or can readily ascertainwithout undue experimentation, specific polynucleotide sequences whichcan be inserted into the retroviral genome to target a specificretroviral vector containing the CARM1 sense or antisensepolynucleotide.

Since recombinant retroviral vectors usually are defective, they requireassistance to produce infectious vector particles. This assistance canbe provided, for example, by using helper cell lines that containplasmids encoding all of the structural genes of the retrovirus underthe control of regulatory sequences within the LTR. These plasmids aremissing a nucleotide sequence which enables the packaging mechanism torecognize an RNA transcript for encapsidation. Helper cell lines whichhave deletions of the packaging signal include but are not limited toPSI.2, PA317 and PA12, for example. These cell lines produce emptyvirions, since no genome is packaged. If a retroviral vector in whichthe packaging signal is intact, but the structural genes are replaced byother genes of interest is introduced into such cells, the vector willbe packaged and vector virions produced.

Since CARM1 promotes the action of nuclear receptors, CARM1 or vectorsexpressing CARM1 may be useful as agonists to stimulate processesmediated by nuclear receptors. For example, glucocorticoids are used asanti-inflammatory agents. Gene therapy applications of CARM1 may enhancethe anti-inflammatory effects of glucocorticoids and could thus enhancethe glucocoticoids' therapeutic effectiveness or reduce theconcentration of glucocorticoids required to provide the desiredanti-inflammatory effects.

The CARM1 nucleotide and predicted amino acid sequence, combined withthe functional domains of CARM1, can be used to design modified forms ofCARM1 that lack the methyltransferase activity but retain the ability tobind GRIP1-type coactivators. For example, we have shown that mutationsin the region of CARM1 that contains the methyltransferase activityproduce such a modified CARM1 protein. Also, a fragment of CARM1 proteinthat contains the GRIP1-binding function but lacks the methyltransferaseregion will also have the same properties. Such forms of CARM1 have a“dominant negative” effect on nuclear receptor function; i.e., whenexpressed in cells, these dominant negative forms of CARM1 reduce theactivity of nuclear receptors. This approach is effective in cells thatnaturally express CARM1 or a functionally equivalent protein from thenative endogenous gene. The dominant negative variant of CARM1interferes with the function of the endogenous CARM1 (or functionallyequivalent protein) as follows: When nuclear receptors bind to a targetgene, they recruit a GRIP1-type coactivator, which would normallyrecruit CARM1. However, if the dominant negative form of CARM1 isexpressed in higher levels than the endogenous intact CARM1, then thedominant negative CARM1 is more likely to bind to GRIP1 instead of theendogenous active CARM1. The recruited dominant negative form of CARM1fails to activate gene expression (since it has no methyltransferase),and also blocks the endogenous intact CARM1 protein from binding toGRIP1 and carrying out its function. Thus, the expression of thedominant negative CARM1 reduces the nuclear receptor's ability toactivate gene expression by interfering with the function of endogenousCARM1. The same forms of CARM1 should have a dominant negative effect onany transcription factor whose function is normally enhanced by intactCARM1. We have demonstrated that a CARM1 mutant (CARM1 VLD mutant), inwhich the amino acids valine189, leucine190, and aspartic acid191(V189A/L190A/D191A) have all been changed to alanine, lacksmethyltransferase activity, lacks coactivator activity, and inhibitsnuclear receptor function in conditions where GRIP1-type coactivatorsare limiting.

Examples of specific uses for such antagonistic reagents are in thetreatment of breast cancer and prostate cancer. Most breast cancers, atleast initially, rely on estrogen for growth; and most prostate cancers,at least initially, depend on androgens for growth. Since CARM1 promotesestrogen and androgen receptor action, antagonists of CARM1 or othermethyltransferases may block or partially block the growth promotingeffects of the hormones estrogen and androgen on these tumors. Theseantagonists may serve as effective chemotherapeutic agents, either whenused alone or when used in combination with other types of treatments.

Polypeptides of the Invention

The isolated polypeptides of the invention include, but are not limitedto, a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 orfragments thereof.

The invention also relates to methods for producing a polypeptidecomprising growing a culture of the cells of the invention in a suitableculture medium, and purifying the protein from the culture. For example,the methods of the invention include a process for producing apolypeptide in which a host cell containing a suitable expression vectorthat includes a polynucleotide of the invention is cultured underconditions that allow expression of the encoded polypeptide. Thepolypeptide can be recovered from the culture, conveniently from theculture medium, and further purified. Preferred embodiments includethose in which the protein produced by such process is a full length ormature form of the protein.

The invention also provides a polypeptide including an amino acidsequence that is substantially equivalent to SEQ ID NO: 2. Polypeptidesaccording to the invention can have at least about 80%, and moretypically at least about 90%, and even more typically 95 sequenceidentity to SEQ ID NO: 2.

The present invention further provides isolated polypeptides encoded bythe nucleic acid fragments of the present invention or by degeneratevariants of the nucleic acid fragments of the present invention. By“degenerate variant” is intended nucleotide fragments which differ froma nucleic acid fragment of the present invention (e.g., an ORF) bynucleotide sequence but, due to the degeneracy of the genetic code,encode an identical polypeptide sequence. Preferred nucleic acidfragments of the present invention are the ORFs that encode proteins.

Methodologies known in the art can be utilized to obtain any one of theisolated polypeptides or proteins of the present invention. At thesimplest level, the amino acid sequence can be synthesized usingcommercially available peptide synthesizers. This is particularly usefulin producing small peptides and fragments of larger polypeptides.Fragments are useful, for example, in generating antibodies against thenative polypeptide. In an alternative method, the polypeptide or proteinis purified from cells which naturally produce the polypeptide orprotein. One skilled in the art can readily follow known methods forisolating polypeptides and proteins to obtain one of the isolatedpolypeptides or proteins of the present invention. These include, butare not limited to, immunochromatography, HPLC, size-exclusionchromatography, ion-exchange chromatography, and immuno-affinitychromatography. See, e.g., Scopes, Protein Purification: Principles andPractice, Springer-Verlag (1994); Sambrook, et al., in MolecularCloning: A Laboratory Manual (supra); Ausubel et al., Current Protocolsin Molecular Biology (supra).

The polypeptides and proteins of the present invention can alternativelybe purified from cells which have been altered to express the desiredpolypeptide or protein. One skilled in the art can readily adaptprocedures for introducing and expressing either recombinant orsynthetic sequences into eukaryotic or prokaryotic cells in order togenerate a cell which produces one of the polypeptides or proteins ofthe present invention. The purified polypeptides can be used in in vitrobinding assays which are well known in the art to identify moleculeswhich bind to the polypeptides.

The protein may also be produced by known conventional chemicalsynthesis. Methods for constructing the proteins of the presentinvention by synthetic means are known to those skilled in the art. Forpolypeptides more than about 100 amino acid residues, a number ofsmaller peptides will be chemically synthesized and ligated eitherchemically or enzymatically to provide the desired full-lengthpolypeptide. The synthetically-constructed protein sequences, by virtueof sharing primary, secondary or tertiary structural and/orconformational characteristics with naturally occurring proteins maypossess biological properties in common therewith. Thus, they may beemployed as biologically active or immunological substitutes fornatural, purified proteins in screening of therapeutic compounds and inimmunological processes for the development of antibodies.

The proteins provided herein also include proteins characterized byamino acid sequences substantially equivalent to those of purifiedproteins but into which modification are naturally provided ordeliberately engineered. For example, modifications in the peptide orDNA sequences can be made by those skilled in the art using knowntechniques. Modifications of interest in the protein sequences mayinclude the alteration, substitution, replacement, insertion or deletionof a selected amino acid residue in the coding sequence. For example,one or more of the cysteine residues may be deleted or replaced withanother amino acid to alter the conformation of the molecule. Techniquesfor such alteration, substitution, replacement, insertion or deletionare well known to those skilled in the art (see, e.g., U.S. Pat. No.4,518,584). Preferably, such alteration, substitution, replacement,insertion or deletion retains the desired activity of the protein.

Other fragments and derivatives of the sequences of proteins which wouldbe expected to retain protein activity in whole or in part and may thusbe useful for screening or other immunological methodologies may also beeasily made by those skilled in the art given the disclosures herein.Such modifications are intended to be encompassed by the presentinvention.

The protein of the invention may also be expressed in a form that willfacilitate purification. For example, it may be expressed as a fusionprotein, such as those of maltose binding protein (MBP),glutathione-S-transferase (GST) or thioredoxin (TRX). Kits forexpression and purification of such fusion proteins are commerciallyavailable from New England BioLab (Beverly, Mass.), Pharmacia(Piscataway, N.J.) and Invitrogen (Carlsbad, Calif.), respectively. Theprotein also can be tagged with an epitope and subsequently purified byusing a specific antibody directed to such epitope. One such epitope(“Flag”) is commercially available from Kodak (New Haven, Conn.).

Our knowledge of CARM1 should make it possible to design and screendrugs that block the methyltransferase activity of CARM1. The CARM1protein can in principle be used for X-ray crystallographic, or otherstructural studies, to determine the 3 dimensional structure of theactive site (including the binding sites for S-adenosylmethionine andthe protein substrate which accepts methyl groups) of themethyltransferase region of CARM1. Once determined, this structure canbe used for rational drug design, to design drugs to block the substratebinding and activate sites of CARM1. These or randomly selectedcandidates can be screened using the methyltransferase activity assayswe have developed.

There are other protein arginine methyltransferases related to CARM1(Lin, W-J. et al. 1996; Gary, J. D. et al. 1996; Aletta, J. M. et al.1998), and there may be others which are unknown at this time. Some ofthese other protein arginine methyltransferases and possibly even someother types of protein methyltransferases (e.g., lysinemethyltransferases and carboxyl methyltransferases (Aletta, J. M. et al.1998) may also be involved in gene regulation by a mechanism similar tothat of CARM1. Our knowledge of the CARM1 sequence and mechanismprovides the tools to search for related genes and proteins and theknowledge to determine whether any of these other methyltransferases areinvolved in regulation of transcription.

Antibodies

Another aspect of the invention is an antibody that specifically bindsthe polypeptide of the invention. Such antibodies can be eithermonoclonal or polyclonal antibodies, as well as fragments thereof andhumanized forms or fully human forms, such as those produced intransgenic animals. The invention further provides a hybridoma thatproduces an antibody according to the invention. Antibodies of theinvention are useful for detection and/or purification of thepolypeptides of the invention.

Protein of the invention may also be used to immunize animals to obtainpolyclonal and monoclonal antibodies that react specifically with theprotein. Such antibodies may be obtained using either the entire proteinor fragments thereof as an immunogen. The peptide immunogensadditionally may contain a cysteine residue at the amino or carboxylterminus, and are conjugated to a hapten such as keyhole limpethemocyanin (KLH). Methods for synthesizing such peptides are known inthe art, for example, as in R. P. Merrifield, J. Amer. Chem. Soc. 85,2149-2154 (1963); J. L. Krstenansky, et al., FEBS Lett. 211, 10 (1987).Monoclonal antibodies binding to the protein of the invention may beuseful diagnostic agents for the immunodetection of the protein.Neutralizing monoclonal antibodies binding to the protein may also beuseful therapeutics for conditions associated with excess production oraccumulation of the protein. In general, techniques for preparingpolyclonal and monoclonal antibodies as well as hybridomas capable ofproducing the desired antibody are well known in the art (Campbell, A.M., Monoclonal Antibodies Technology: Laboratory Techniques inBiochemistry and Molecular Biolozy, Elsevier Science Publishers,Amsterdam, The Netherlands (1984); St. Groth et al., J. Immunol. 35:1-21(1990); Kohler and Milstein, Nature 256:495-497 (1975)). Other usefultechniques include the trioma technique and the human B-cell hybridomatechnique (Kozbor et al., Immunology Today 4:72 (1983); Cole et al., inMonoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), pp.77-96).

Any animal (rabbit, etc.) which is known to produce antibodies can beimmunized with a peptide or polypeptide of the invention. Methods forimmunization are well known in the art. Such methods includesubcutaneous or intraperitoneal injection of the polypeptide. Oneskilled in the art will recognize that the amount of the protein encodedby the ORF of the present invention used for immunization will varybased on the animal which is immunized, the antigenicity of the peptideand the site of injection. The protein that is used as an immunogen maybe modified or administered with an adjuvant to increase the protein'santigenicity. Methods of increasing the antigenicity of a protein arewell known in the art and include, but are not limited to, coupling theantigen with a heterologous protein (such as globulin orβ-galactosidase) or through the inclusion of an adjuvant duringimmunization.

For monoclonal antibodies, spleen cells from the immunized animals areremoved, fused with myeloma cells, such as SP2/0-Ag14 myeloma cells, andallowed to become monoclonal antibody producing hybridoma cells. Any oneof a number of methods well known in the art can be used to identify thehybridoma cell which produces an antibody with the desiredcharacteristics. These include screening the hybridomas with an ELISAassay, western blot analysis, or radioimmunoassay (Lutz et al., Exp.Cell Research, 175:109-124 (1988)).

Hybridomas secreting the desired antibodies are cloned and the class andsubclass is determined using procedures known in the art (Campbell, A.M., Monoclonal Antibody Technology: Laboratory Techniques inBiochemistry and Molecular Biology, Elsevier Science Publishers,Amsterdam, The Netherlands (1984)). Techniques described for theproduction of single chain antibodies (U.S. Pat. No. 4,946,778) can beadapted to produce single chain antibodies to proteins of the presentinvention.

For polyclonal antibodies, antibody containing antiserum is isolatedfrom the immunized animal and is screened for the presence of antibodieswith the desired specificity using one of the above-describedprocedures. The present invention further provides the above-describedantibodies in detectably labeled form. Antibodies can be detectablylabeled through the use of radioisotopes, affinity labels (such asbiotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase,alkaline phosphatase, etc.) fluorescent labels (such as FITC orrhodamine, etc.), paramagnetic atoms, etc. Procedures for accomplishingsuch labeling are well-known in the art, for example, see Stemberger, L.A. et al., J. Histochem. Cytochem. 18:315 (1970); Bayer, E. A. et al.,Meth. Enzym. 62:308 (1979); Engval, E. et al., Immunol. 109:129 (1972);Goding, J. W. J. Immunol. Meth. 13:215 (1976).

In diagnostic uses, it is possible that some medical conditions mayderive from abnormal forms or levels of expression of CARM1 or othermethyltransferases. Thus, nucleic acid and antibody reagents derivedfrom CARM1 or other methyltransferases may be used to screen humans forsuch abnormalities. Similarly, there may be different alleles of CARM1or other methyltransferases that predispose carriers to be moresusceptible to specific drugs or diseases. The CARM1 reagents can beused to define such allelic variations and subsequently to screen forthem.

Antibodies of the invention can also be generated that specificallyrecognize substrates that have been methylated by CARM1. For example,CARM1 methylates residues arg2, arg17 and arg26 of histone H3.Antibodies to peptides containing methylated arginines at these, orother positions of CARM1 methylation, are useful for studying the roleof methylation in gene expression.

Methyltransferase Activity

CARM1 can transfer one or more methyl groups from S-adenosylmethionineto an arginine residue in proteins and in synthetic peptides.Appropriate substrate proteins include histones. CARM1 can transfermethyl groups from S-adenosylmethionine to one or more arginine residuesin histone H3, producing monomethyl and asymmetrically dimethylatedN^(G)N^(G)-dimethylarginine residues in histone H3. The CARM1 VLD (SEQID NO:3) mutant lacks methyltransferase activity for both histone H3 andthe synthetic peptide substrates and lacks coactivator activity.

Substrates of CARM1

While the identity of additional proteins (other than histone H3) thatCARM1 methylates remains unknown, we have established a procedure foridentifying proteins that are methylated by CARM1, by incubatingcandidate substrate proteins or protein fractions or extracts withrecombinant CARM1 and S-adenosylmethionine and then analyzing theproducts by chromatography or electrophoresis. The purified protein canbe sequenced to learn its identity. The yeast two hybrid system, used todiscover CARM1, also should be useful for defining proteins that bind toCARM1 methyltransferase and thus are possible substrates. Onceidentified, these methylation substrates of CARM1 should be useful asreagents for studying the role and mechanism of methylation in generegulation. They also serve as additional sites of intervention forblocking or enhancing gene expression. This is accomplished byincreasing the expression of the protein substrate or by reducingexpression of the protein substrate, for example by using antisensetechniques or by expressing altered forms of the protein substrate whichhave a dominant negative effect and thus block the function of theendogenous native protein substrate.

Because protein methylation is involved in regulation of genetranscription, a mechanism for demethylation of the same proteins likelyexists. Histone H3 is a substrate for CARM1, and we have describedmethods for identifying other protein substrates of CARM1 above. Thesemethylated proteins can serve as the basis for identifying demethylatingenzymes. In such a method, a methylated protein preparation is incubatedwith cell extracts, fractions of cell extracts or with candidateproteins. Demethylation can be monitored by release of radioactivity ifthe methylated protein is prepared with radioactively labeledS-adenosyl-methionine. Demethylation can also be monitored bychromatographic changes, using techniques such as ion-exchangechromatography; by mass spectrometry; by spectroscopic techniques suchas fluorescence spectropscopy; or by immunoassays with antibodies raisedagainst the methylated or non-methylated forms of the protein. Onceidentified, these demethylating enzymes can be used as the basis fordeveloping reagents to enhance or block demethylation. Blocking orenhancing demethylation should have the opposite effect from blocking orenhancing methylation by CARM1.

Screening of CARM1 Inhibitors

Inhibitors of CARM1 can be discovered using the methods of the inventionthat act through a variety of mechanisms. In one embodiment, moleculesare screened for their ability to inhibit CARM1 methyltransferaseactivity. Methyltransferase activity can be determined using any of theassays described herein, or other suitable biochemical assays. Forexample, in one embodiment a substrate protein, such as histone H3 isincubated with a candidate inhibitor molecule or pool of molecules alongwith CARM1 (SEQ ID NO: 2) and radioactively labeledS-adenosylmethionine. The degree of radioactive labeling of the targethistone is measured by separating the labeled protein from the freeS-adenosylmethionine. Separation may be effected by chromatography or byusing a low molecular weight cut-off membrane, through which the freeS-adenosylmethionine passes, but the labeled protein is retarded. Theactivity of CARM1 is then compared in the presence and absence of thecandidate inhibitor.

CARM1 inhibitors can also be discovered that prevent interaction ofCARM1 with a coactivator such as GRIP1. The disclosed two-hybrid assaysfor measuring the binding interaction between coactivators and CARM1 arealso suitable for use as a screening system to identify compounds thatcan block binding of CARM1 to GRIP1-type coactivators.

In one embodiment, CARM1 (SEQ ID NO: 1) or a fragment thereof, isexpressed in a host cell as a fusion with either a DNA binding domain(DBD) or with a transcriptional activation domain (AD). DNA bindingdomains are well known in the art, and can be chosen from any DNAbinding protein or transcription factor. In one embodiment, CARM1 isexpressed fused with the DNA binding domain of Gal4. In anotherembodiment, CARM1 is expressed instead fused to a transcriptionalactivation domain from Gal4.

A GRIP1-type coactivator, or a fragment thereof, is expressed as afusion with either a DNA binding domain or with a transcriptionalactivation domain, but not with the domain type chosen for CARM1. IfCARM1 is fused with a DNA binding domain, then the GRIP1-typecoactivator domain must be fused with a transcriptional activatingdomain.

In such a method, a reporter gene construct is also provided. Thereporter gene construct comprises a reporter gene and a promoter region.Reporter genes encode a protein that can be directly observed or can beindirectly observed through an enzymatic activity or through immunogenicdetection methods. Directly observable proteins can be fluorescentproteins, such as the green fluorescent protein (GFP) of Aequorea.Indirectly observable proteins commonly possess an enzymatic activitycapable of affecting a chromogenic or fluorogenic change in a specificsubstrate. Such proteins include P-lactamase, luciferase andβ-galactosidase. Reporter gene expression can also be monitored withantibodies directed towards the gene product, or by measuring the RNAlevels produced.

In the two hybrid system, the interaction of CARM1-AD hybrid proteinwith the GRIP1-DBD hybrid protein leads to the expression of thereporter gene, and thus, the expression of the reporter gene serves asan indication that CARM1 and GRIP1 can bind to each other.

Upon consideration of the present disclosure, one of skill in the artwill appreciate that many other embodiments and variations may be madein the scope of the present invention. Accordingly, it is intended thatthe broader aspects of the present invention not be limited to thedisclosure of the following examples, but rather only to the scope ofthe appended claims.

EXAMPLE 1

Isolation of murine CARM1 cDNA

A 3.2-kb partial CARM1 cDNA clone with an open reading frame of 606amino acids (CARM1(3-608)), followed by a 1.4 kb 3′-untranslated regionand a poly A sequence, was isolated from a mouse 17-day embryo libraryby using the yeast two-hybrid system as described previously (Hong, H.et al. 1996). The EcoRI library (Clontech) was in vector pGAD 10 whichhas a leu2 marker gene; the bait was GRIP1_(c) (GRIP (1122-1462)) invector pGBT9 (Clontech) which has a trpl marker gene. Further screeningof a lambdaphage library of mouse 11-day embryo cDNA clones (Stratagene)identified additional 5′-sequences and allowed construction of aputative full length coding region for CARM1 (608 amino acids). Aminoacids 143-457 of CARM1 share 30% identity with hPRMT1 and yODP1. A clonecoding for a C-terminal fragment of α-actinin was isolated in the sameyeast two hybrid screen with pGBT9.GRIP1_(c).

A BLAST search of the GenBank database (Altschul, S. F. et al. (1990) J.Mol. Biol. 215:40314 410) indicated that this coding region represents anovel protein, whose central region shares extensive homology with afamily of proteins with arginine-specific protein methyltransferaseactivity (FIG. 1). We therefore named the new protein CoactivatorAssociated arginine (R) Methyltransferase 1 (CARM1). RNA blot analysisindicated that the CARM1 cDNA represents a 3.8-kb mRNA which isexpressed widely, but not evenly, in adult mouse tissues including inheart, brain, liver, kidney, and testis; testis also contains ahomologous 4.1-kb RNA species (FIG. 2). Lower expression was observed inspleen, lung, and skeletal muscle. Northern blot analysis was performedas shown in FIG. 2 with a 0.6-kb BamHI cDNA fragment (representing CARM1codons 3-198) and with RNA from multiple tissues as described previously(Hong et al. 1997).

EXAMPLE 2

Construction of Plasmids

Mammalian cell expression vector: pSG5.HA was constructed by inserting asynthetic sequence coding for a translation start signal, HA tag, EcoRIsite, and XhoI site into the EcoRI-BamHI site of pSG5 (Stratagene),which has SV40 and T7 promoters. The original EcoRI site is destroyed bythis insertion, but the BamHI site is preserved, leaving a multiplecloning site after the HA tag containing EcoRI, XhoI, BamHI, and BglIIsites. The following protein coding regions were cloned into pSG5.HA, inframe with the HA tag, using the indicated insertion sites: GRIP1(5-1462) (full length) and CARM1 (3-608) (full length) at the EcoRIsite; GRIP1 (5-765) at the EcoRl-XhoI site; GRIP1 (730-1121) and GRIP1(1121-1462) were EcoRI-SalI fragments inserted at the EcoRI-XhoI site;SRC-1a (1-1441) (full length) was a SmaI-SalI fragment inserted at theEcoRI site, which was blunted by filling with Klenow polymerase, and theXhoI site. Expression vector for Gal4DBD-GRIP1_(c) was constructed byinserting an EcoRl-BglII fragment coding for GRIP1 (1122-1462) into pM(Clontech). Vectors for GST fusion proteins were constructed in pGEX-4T1Pharmacia): for GST-CARM1 the original 3.2-kb EcoRI fragment frompGAD10.CARM1 was inserted; for GST-GRP1c (amino acids 1122-1462) aEcoRI-SalI fragment was inserted. Yeast expression vectors for Gal4DBDfused to various GRIP1 fragments were constructed by insertingEcoRI-SalI fragments into pGBT9. The GRIP1_(c)Δ19 and CARM1 VLDmutations were engineered with the Promega Gene Editor Kit.Constructions of all the above plasmids was described previously (Chen,D. et al. (1999) Science 284:2174-2177).

EXAMPLE 3

Binding Interactions of CARM1

This example demonstrates that CARM1 interacts with GRIP1. The bindingof GRIP1_(c) to CARM1 observed in the yeast two-hybrid system wasconfirmed in vitro, by incubating glutathione S-transferase (GST) fusionproteins attached to glutathione agarose beads with labeled proteins orprotein fragments translated in vitro. GST-CARM1 bound GRIP1_(c) (aminoacids 1122-1462) but not protein fragments representing GRIP1 aminoacids 5-765 or 730-1121 (FIG. 3A). Conversely, GST-GRIP1_(c) bound CARM1and the VLD to AAA mutant of CARM1 (FIG. 3B). GST-CARM1 not only boundGRIP1 but also the other two members of the p160 coactivator family,SRC-1a and ACTR (FIG. 3A). Thus, FIG. 3 shows the binding of CARM1 tothe C-terminal region of p160 coactivators. GST fusion proteins of CARM1or the indicated GRIP1 fragments produced in E. coli strain BL21(Stratagene), were bound to glutathione-agarose beads and incubated withlabeled full length CARM1 or p160 coactivators or GRIP1 fragmentstranslated in vitro from vector pSG5.HA-CARM1, pSG5.HA-GRIP1,pSG5.HA-SRC-1a (Chen, D. et al. 1999), or pCMX.ACTR (Chen et al. 1997);bound labeled proteins were eluted and analyzed by SDS polyacrylamidegel electrophoresis as described previously (Hong et al. 1996). A mutantform of CARM1 with the triple amino acid substitution (VLD changed toAAA) shown in FIG. 1 still retains the ability to bind to the C-terminalfragment of GRIP1.

The binding site for CARM1 in GRIP1_(c) was further mapped by using theyeast two hybrid system. When GRIP1_(c) was bisected between amino acids1210 and 1211, the N-terminal fragment fails to bind CARM1, while theC-terminal fragment retains binding activity; thus GRIP1211-1462 issufficient for CARM1 binding while amino acids 1121-1210 are neithernecessary nor sufficient (FIG. 4). When GRIP1_(c) was bisected betweenamino acids 1305 and 1306, neither fragment bound CARM1, indicating thatsequences near this boundary were important for CARM1 binding. Thisconclusion was supported by the finding that deletion of amino acids1291-1309 (GRIP1_(c)Δ19 mutant), which are highly conserved among p160proteins (Anzick, S. L. et al. 1997), eliminate CARM1 binding. Thesmallest GRIP1 fragment that binds to CARM1 is the fragment from1211-1350. Controls with α-actinin, another protein found to bindGRIP1_(c) in the yeast two hybrid screen, had a different pattern ofbinding to the GRIP1 fragments and provided positive and negativecontrols. We conclude that CARM1 binds to the C-terminal region of GRIP1defined by amino acids 1211-1350 and that a highly conserved stretch of19 amino acids (1291-1309) is important for CARM1 binding.

EXAMPLE 4

Enhancement of GRIP1 and NR Function by Secondary Coactivator CARM1

This example demonstrates that CARM1 expression in mammalian cellsenhances the transcriptional activation activity of GRIP1_(c) fused tothe DBD of Gal4 protein. In transient transfections of CV-1 cells,Gal4DBD-GRIP1_(c) weakly activates expression of a reporter gene with apromoter containing Gal4 binding sites; co-expression of CARM1 enhancesreporter gene activity in a dose-dependent manner and provides a maximumstimulation of more than 10-fold (FIG. 5). CARM1 expression has littleif any effect on the activity of Gal4DBD alone (FIG. 6A). CARM1 alsoenhances the activity of full length GRIP1 fused to Gal4DBD.

CARM1 also enhances GRIP1's coactivator function for nuclear receptors(NR). are When the androgen receptor, estrogen receptor, and thyroidhormone receptor are expressed in CV-1 cells by transient transfection,their abilities to activate transcription of a reporter gene carryingappropriate hormone response elements in the promoter are hormonedependent (FIG. 6B, lanes a & b). Co-expression of GRIP1 from aco-transfected plasmid causes a 2 to 27-fold enhancement of reportergene expression by the hormone-activated NR (lane d). These activitiesare enhanced 2 to 4-fold more by co-expression of CARM1 with the NR andGRIP1 (lane e). However, in the absence of exogenous GRIP1, CARM1 haslittle or no effect on the activity of the NR (lane c). Co-expression ofNR, GRIP1, and CARM1 in the absence of hormone produces extremely lowreporter gene activities equivalent to those seen with NR alone in theabsence of hormone (lane f). A similar enhancement of NR function byCARM1 is observed when SRC-1a or ACTR (two other GREPI relatedcoactivators) is substituted for GRIP1 in a similar experiment. The factthat CARM1's ability to enhance NR activity depends on co-expression ofexogenous GRIP1 is consistent with a model whereby CARM1 interacts withNRs indirectly, through a p160 coactivator, rather than directly (FIG.7). It also suggests that in the transient transfection assays, theexpression of exogenous NRs renders the levels of endogenous p160coactivators limiting, so that the effects of exogenous CARM1 expressioncan only be observed when additional p160 coactivators are alsoexpressed. We conclude that CARM1 acts as a secondary coactivator forNRs by binding to and mediating or enhancing the activity of the p160primary coactivators.

EXAMPLE 5

Histone Methyltransferase Activity of CARM1

This example shows that CARM1 is a protein arginine methyltransferase.The homology between CARM1 and arginine-specific proteinmethyltransferases includes sequences that are highly conservedthroughout the family and are believed to be important formethyltransferase activity (FIG. 1). We compared the methyltransferaseactivities of GST fusion proteins of CARM1 and a related mammalianenzyme, Protein arginine (R) Methyltransferase 1 (PRMT1) (Lin, W-J. etal. 1996), for various substrates, using S-adenosylmethionine labeled inthe donor methyl group. Mixed histones are good substrates for bothenzymes (FIG. 8). Gel electrophoresis and autoradiography of themethylated histone products, and tests with purified individual histonespecies, indicate that CARM1 methylates histones H3 and H2a, while PRMT1methylates histones H4 and H2a (FIG. 8B). Both enzymes methylate histone2a in the absence of other histones but not in the histone mixture,suggesting that hetero-oligomerization of the histones may renderhistone 2a inaccessible to methylation (FIG. 8B). The positions of thesmall amounts of labeled products in the histone H2b lanes for CARM1 andPRMT1 suggest that these products are minor amounts of H3 and H4contaminating the H2b preparation. The specific activities of CARM1 andPRMT1 with the mixed histone substrate are very similar (Table 1). Ourresult for PRMT1 is different from one in a previous report (Gary andClarke 1998), that PRMT1 methylates histone H2b but none of the othercore histones. RNA binding protein hnRNPA1 is a good substrate forPRMT1, as shown previously (Lin W-J, et al. 1996), but is not methylatedby CARM1 (FIG. 8C). Both enzymes methylate the glycine-rich R1 peptidesubstrate (SEQ ID NO: 4: GGFGGRGGFG-NH₂), which was previously shown tobe a good substrate for PRMT1 and other protein argininemethyltransferases (Lin, W-J. et al. 1996; Najbauer, J. et al. 1993).However, this peptide is a relatively poor substrate for CARM1; thespecific activity of GST-PRMT1 for the R1 peptide is approximately 100times higher than that of GST-CARM1 (Table 1). CARM1 fails to methylatethe same peptide with lysine substituted for the arginine residue,demonstrating its specificity for arginine.

TABLE 1 Relative methyltransferase activities of GST-CARM1 andGST-PRMT1. Methyltransferase reactions (50 μl) were carried out asdescribed in FIG. 8 at enzyme concentrations of 0.03-0.05 mg/ml.Reactions were stopped by addition of 25 μl of 1.5% (v/v)trifluoroacetic acid (TFA), 15% (v/v) acetonitrile in water andsubjected to reversed-phase HPLC as described (Najbauer et al. 1993) toseparate the substrate from unreacted S-adenosylmethionine. For thehistone methylation, TFA in the HPLC solvents was increased to 0.3%(v/v) and the gradient was modified to accommodate the more retentivebehavior of the histones Methyltransferase specific activity(pmol/min/mg) Substrate GST-CARM1 GST-PRMT1 R1 peptide (120 μM)21.6-54.5^(a) 3,070 SEQ ID NO: 4 GGFGGRGGFG-NH₂ K1 peptide (120 μM) 0.7not determined SEQ ID NO: 5 GGFGGKGGFG-NH₂ mixed histones (2.7 mg/ml)971 1,180 (calf thymus) ^(a)Result of two separate determinations usingdifferent preparations of GST-CARM1.

EXAMPLE 6

Identification of the Methylated Amino Acids Produced in Histone H3 byCARM1

Histone H3 was incubated for 60 min at 30° C. in a 100 μl methylationreaction as described in FIG. 8, containing 0.024 mg/ml GST-CARM1 and0.63 mg/ml H3. The reaction was stopped with 25 μl of 3% (v/v)trifluoroacetic acid (TFA), 15% (v/v) acetonitrile, and 100 μl wasinjected into a 3 cm×4.6 mm RP-300 reversed-phase guard column (PerkinElmer-Brownlee) equilibrated with 80% solvent A (0.3% TFA in water) and20% solvent B (0.3% TFA in acetonitrile). Methylated H3 was separatedfrom unreacted S-adenosylmethionine using a gradient of 20-80% solvent Bover 5 min at a flow rate of 1.0 ml/min. H3 eluted as a broad complexpeak detected by monitoring absorbance at 214 nm. The H3 pool wasreduced to dryness in a vacuum centrifuge and then subjected to acidhydrolysis in 6 N HCl at 112 ° C. for 20 h. A portion of the hydrolyzatewas derivatized with o-phthaldialdehyde (Jones, B. N., Methods ofProtein Microcharacterization, J. E. Shively, Ed. (Humana Press,Clifton, N.J., 1986), p. 337) and injected into a 10 cm×4.6 mm RaininMicrosorb 800PA-C3 column fitted with a guard module and equilibratedwith 95% solvent A (50 mM Na-acetate, pH 5.9: methanol: tetrahydrofuran,79:20:1) and 5% solvent B (50 mM Na-acetate, pH 5.9:methanol, 20:80)Elution was carried out with a linear gradient of 5-40% B over 20 min ata flow rate of 1.0 ml/min. Radioactivity in the fractions was determinedby liquid scintillation counting, and peak identity was determined bycomparison to derivatized standards including the three major forms ofmethylarginine and methyllysine. In addition, another portion of theacid hydrolyzate was subjected to ascending chromatography on thinlayers on cellulose using pyridine: acetone: ammonium hydroxide:water(15:9:1.5:6) (Desrosiers, R. and Tanguay (1988) J. Biol. Chem.263:4686). Radioactive spots corresponding to the positions of the threeforms of methylarginine (which all separated from each other) wereremoved by scraping the chromatogram, and quantified by liquidscintillation counting. Sources of standards: monomethyl-L-arginine andtrimethyl-L-lysine, Calbiochem; N,N′-dimethyl-Larginine andmonomethyl-L-lysine, Sigma; N,N-dimethyl-L-arginine, Chemical Dynamics,Corp.; dimethyl-L-lysine, Serva.

When histone H3 is methylated by CARM1, hydrolyzed to amino acids,derivatized, and analyzed by high performance liquid chromatography (asdescribed above), all of the radioactivity from histone H3 co-elutes ina single peak along with the derivatized standards ofN^(G)-monomethylarginine and N^(G),N^(G)dimethylarginine (which did notseparate from each other). The radioactive peak was well separated fromstandards of N^(G),N′^(G)-dimethylarginine, N^(ε)-monomethyllysine,N^(ε)-dimethyllysine, and N^(ε)-triethyllysine. On thin layerchromatography of the hydrolyzate, approximately 70% of the radiolabelmigrated with N^(G),N^(G)-dimethylarginine (asymmetrically dimethylatedin the guanidino group) and the remaining 30% withN^(G)-monomethylarginine. In confirmation of the HPLC results, nosignificant label migrated with N^(G),N^(G)-dimethylarginine(symmetrically dimethylated in the guanidino group). Methylation ofmixed histones by PRMT1 was previously shown to produce the same typesof methylated arginine residues (Gary and Clark 1998). However, whilethey produce the same types of methylated arginine residues, CARM1 andPRMT1 have dramatically different protein substrate specificities (FIG.8 and Table 1). Histone H4, nucleolin, fibrillarin and hnRNPA1, as wellas the peptide substrate, all have arginine-containing glycine-richmotifs, whereas histone H3 does not (Najbauer, J. et al. 1993; Lin, W-J.et al. 1996; Genbank Accession Numbers, for calf thymus histone H3,70749, and for histone H4, 70762). Thus, it appears that PRMT1 prefersto methylate arginines found in the glycine-rich motifs, whereas CARM1targets a different arginine-containing motif in proteins.

EXAMPLE 7

Sites of CARM1 Methylation of Histone H3

CARM1 methylated the following residues of histone H3, as determined bymass spectrometry analysis: arg2 (minor), arg17 (major), arg26 (major),and one or more of the 4 arginine residues within the histone H3 peptideregion comprising residues 128-134. N-terminal sequencing of histone H3labeled by CARM1-mediated methylation confirmed that within the first 20amino acids of histone H3, arg2 was a minor methylation site, arg17 wasa major methylation site, and arg8 was not methylated (those are theonly three arg residues within the first 20 amino acids of H3). Thesequencing run was only able to analyze the first 20 amino acids fromthe N-terminus.

EXAMPLE 8

The Role of Methyltransferase Activity in Transcription

This example show that CARM1's methyltransferase activity is necessaryfor its activity as a coactivator of transcription. We made a mutationin the CARM1 coding sequences that resulted in replacement of threeamino acids, valine 189, leucine 190, and aspartic acid 191, withalanines. This VLD sequence is located in the region that is most highlyconserved among different members of the protein argininemethyltransferase family (FIG. 1) and is believed to be important forS-adenosylmethionine binding and thus for methyltransferase activity(Lin, W-J. et al. (1996) J. Biol. Chem. 271:15034-15044). This mutationcompletely eliminates the ability of the GST-CARM1 fusion protein tomethylate mixed histones (FIG. 8C) and peptide substrate R1 (SEQ IDNO:4). The same mutation essentially eliminates CARM1's ability toenhance transcriptional activation by a Gal4DBD-GRIP1_(c) fusion protein(FIG. 6A) or by the estrogen receptor (FIG. 6B). Immunoblots oftransfected COS7 cell extracts indicated that both wild type and mutantCARM1 were expressed at similar levels. The VLD mutant retains theability to bind the C-terminal region of GRIP1 (FIG. 3B). The correlatedloss of the methyltransferase activity and coactivator activity of CARM1indicates that methyltransferase activity is important for CARM1 'scoactivator function.

EXAMPLE 9

Synergy of CARM1 with Histone Acetyl Transferase and Other ProteinArginine Methyltransferases in Transcriptional Activation

This example shows that CARM1 and other protein argininemethyltransferases synergistically activate transcription with eachother and with histone acetyl transferases. As shown in FIGS. 9, 10 and11, cells were transiently transfected with combinations of plasmidsencoding GRIP1, CARM1, p300, PRMT1, PRMT2, and PRMT3. At low levels ofexpression of an appropriate nuclear receptor, in this case estrogenreceptor (ER), a combination of GRIP1, CARM1 and p300 are required foractivation of the ER-dependent receptor gene FIG. 10B, left side).Similar effects are observed if PRMT1 is substituted for CARM1, otherp160 coactivators are substituted for GRIP1, or CBP or P/CAF issubstituted for p300. P300, CBP, and P/CAF all have histoneacetyltransferase activity. This indicated that histonemethyltransferases and histone acetyltransferases have cooperative orsynergistic coactivator activity and suggests that methylation andacetylation of histones and/or other proteins in the transcriptioncomplex are cooperative processes in the activation of transcription

The activity of CARM1 is also synergistic with those of PRMT1, PRMT2 orPRMT3. In cells expressing low levels of the orphan (i.e. no ligand)receptor, ERR1 or ERR3, cotransfection of cells with plasmids encodingGRIP1, CARM1 and PRMT1 results in highly increased reporter geneexpression as shown in FIG. 10A. The synergy between CARM1 and PRMT1 isalso observed with three other nuclear receptors: estrogen, androgen,and thyroid hormone receptors (FIG. 9). FIG. 11 shows that CARM1 alsoacts synergistically with either PRMT2 or PRMT3.

Furthermore, due to the very high degree of dependence of the reportergene activity on the presence of CARM1 and/or PRMT1, these conditionsmay prove useful for screening for inhibitors of the methyltransferaseactivity or the coactivator activity associated with thesemethyltransferases. Such transiently transfected cells when they containlow levels of a nuclear receptor will express the nuclearreceptor-dependent reporter gene, in this case luciferase, only in thepresence of GRIP1, CARM1 and p300. Molecules that inhibit either theenzymatic activities of these coactivators or the protein-proteininteractions of these coactivators would reduce the level of signal fromthe reporter gene.

EXAMPLE 10

Anti-CARM1 Antibody

The peptide SEQ ID NO: 6: (C)SPMSIPTNTMHYGS-COOH, representing theC-terminal CARM1 amino acid residues 595-608 was coupled to KLH andinjected into rabbits. The (C) is not part of the CARM1 sequence but wasadded for coupling to KUH. The antiserum was tested at a dilution of1:2000 in western blotting. The positive control was CARM1 translated invitro with no radioactive amino acids; the negative control was aparallel in vitro translation reaction with no CARM1 mRNA. Products fromthese two reactions were separated by molecular weight bySDS-polyacrylamide gel electrophoresis, and the proteins weretransferred from the gel to a nylon membrane. The membrane was incubatedwith the CARM1 antiserum, a secondary HRP-coupled antibody, andvisualized by luminescence. The positive control gave a very strong bandat the expected size for CARM1, while the negative control gave no bandat that position.

All of the publications which are cited within the body of the instantspecification are hereby incorporated by reference in their entirety.

TABLE 2 SEQ ID NO:1. M. musculuc cDNA for CARM1 (GenBank Accession No.AF117887). 1 agggggcctg gagccggacc taagatggca gcggcggcag cgacggcggtggggccgggt 61 gcggggagcg ctggggtggc gggcccgggc ggcgcggggc cctgcgctacagtgtctgtg 121 ttcccgggcg cccgcctcct cactatcggc gacgcgaacg gcgagatccagcggcacgcg 181 gagcagcagg cgctgcgcct tgaggtgcgc gccggaccag acgcggcgggcatcgccctc 241 tacagccatg aagatgtgtg tgttttcaag tgctcggtgt cccgagagacagagtgcagt 301 cgtgtgggca gacagtcctt catcatcacc ctgggctgca acagcgtcctcatccagttt 361 gccacacccc acgatttctg ttctttctac aacatcctga aaacctgtcggggccacaca 421 ctggagcgct ctgtgttcag tgagcggaca gaggaatcct cagctgtgcagtacttccag 481 ttctatggct acctatccca gcagcagaac atgatgcagg actatgtgcggacaggcacc 541 taccagcgtg cgatcctgca gaaccacacg gacttcaagg acaagatcgttctagatgtg 601 ggctgtggct ctgggatcct gtcatttttt gctgctcaag caggagccaggaaaatttat 661 gcagtggaag ccagcaccat ggctcagcat gcagaggtcc tggtgaagagtaacaatctg 721 acagaccgca tcgtggtcat ccctggcaaa gtagaggagg tctcattgcctgagcaagtg 781 gacattatca tctcagagcc catgggctac atgctcttca atgaacgaatgctcgagagc 841 tacctccatg ccaaaaagta cctgaagcct agtggaaaca tgttccccaccattggtgat 901 gtccacctcg cacccttcac tgatgaacag ctctacatgg agcagttcaccaaagccaac 961 ttccggtacc agccatcctt ccatggagtg gacctgtcgg ccctcagaggtgccgctgtg 1021 gatgagtact tccggcaacc tgtggtggac acatttgaca tccggatcctgatggccaaa 1081 tctgtcaagt acacagtgaa cttcttagaa gccaaagaag gcgatttgcacaggatagaa 1141 atcccattca aattccacat gctgcattca gggctagtcc atggcttggccttctggttc 1201 gatgttgctt tcattggctc cataatgacc gtgtggctat ccacagccccaacagagccc 1261 ctgacccact ggtaccaggt ccggtgcctc ttccagtcac cgttgtttgccaaggccggg 1321 gacacgctct cagggacatg tctgcttatt gccaacaaaa gacagagctatgacatcagt 1381 attgtggcac aggtggacca gacaggctcc aagtccagta acctgctggatctaaagaac 1441 cccttcttca ggtacacagg tacaacccca tcacccccac ctggctcacactacacgtct 1501 ccctcggaga atatgtggaa cacaggaagc acctataatc tcagcagcggggtggctgtg 1561 gctggaatgc ctactgccta cgacctgagc agtgttattg ccggcggctccagtgtgggt 1621 cacaacaacc tgattccctt agctaacaca gggattgtca atcacacccactcccggatg 1681 ggctccataa tgagcacggg cattgtccaa ggctcctcag gtgcccagggaggcggcggt 1741 agctccagtg cccactatgc agtcaacaac cagttcacca tgggtggccctgccatctct 1801 atggcctcgc ccatgtccat cccgaccaac accatgcact atgggagttaggtgcctcca 1861 gccgcgacag cactgcgcac tgacagcacc aggaaaccaa atcaagtccaggcccggcac 1921 agccagtggc tgttccccct tgttctggag aagttgttga acacccggtcacagcctcct 1981 tgctatggga acttggacaa ttttgtacac gatgtcgccg ctgccctcaagtacccccag 2041 cccaaccttt ggtcccgagc gcgtgttgct gccatacttt acatgagatcctgttggggc 2101 agccctcatc ctgttctgta ctctccactc tgacctggct ttgacatctgctggaagagg 2161 caagtcctcc cccaaccccc acagctgcac ctgaccaggc aggaggaggccagcagctgc 2221 caccacagac ctggcagcac ccaccccaca acccgtcctt gcacctcccctcacctgggg 2281 tggcagcaca gccagctgga cctctccttc aactaccagg ccacatggtcaccatgggcg 2341 tgacatgctg ctttttttaa ttttattttt ttacgaaaag aaccagtgtcaacccacaga 2401 ccctctgaga aacccggctg gcgcgccaag ccagcagccc ctgttcctaggcccagaggt 2461 tctaggtgag gggtggccct gtcaagcctt cagagtgggc acagcccctcccaccaaagg 2521 gttcacctca aacttgaatg tacaaaccac ccagctgtcc aaaggcctagtccctacttt 2581 ctgctactgt cctgtcctga gccctgaagg cccccctcca tcaaaagcttgaacaggcag 2641 cccagagtgt gtcaccctgg gctactgggg cagacaagaa acctcaaagatctgtcacac 2701 acacacaagg aaggcgtcct ctcctgatag ctgacatagg cctgtgtgttgcgttcacat 2761 tcatgttcta cttaatcctc tcaagacagc aaccctggga aggagcctcgcagggacctc 2821 cccagacaag aagaaaagca aacaaggaag ggtgattaat aagcacaggcagtttcccct 2881 attcccttac cctagagtcc ccacctgaat ggccacagcc tgccacaggaaccccttggc 2941 aaaggctgga gctgctctgt gccaccctcc tgacctgtca gggaatcacagggccctcag 3001 gcagctggga accaggctct ctcctgtcca tcagtaatac tccttgctcggatggccctc 3061 ccccaccttt atataaattc tctggatcac ctttgcatag aaaataaaagtgtttgcttt 3121 gtaa

TABLE 3 SEQ ID NO: 2. Deduced amino acid sequence of CARM1 (GenBankAccession No. AAD41265). 1 maaaaatavg pgagsagvag pggagpcatv svfpgarlltigdangeiqr haeqqalrle 61 vragpdaagi alyshedvcv fkcsvsrete csrvgrqsfiitlgcnsvli qfatphdfcs 121 fynilktcrg htlersvfse rteessavqy fqfygylsqqqnmmqdyvrt gtyqrailqn 181 htdfkdkivl avgcgsgils ffaaqagark iyaveastmaqhaevivksn nltdrivvip 241 gkveevslpe qvdiiisepm gymlfnerml esylhakkylkpsgnmfpti gdvhlapftd 301 eqlymeqftk anfryqpsfh gvdlsalrga avdeyfrqpvvdtfdirilm aksvkytvnf 361 leakegdlhr ieipfkfhml hsglvhglaf wfdvafigsimtvwlstapt eplthwyqvr 421 clfqsplfak agdtlsgtcl liankrqsyd isivaqvdqtgskssnlldl knpffrytgt 481 tpspppgshy tspsenmwnt gstynlssgv avagmptaydlssviaggss vghnnlipla 541 ntgivnhths rmgsimstgi vqgssgaqgg ggsssahyavnnqftmggpa ismaspmsip 601 tntmhygs

TABLE 4 SEQ ID NO:3. Sequence of CARMl VLD to AAA variant. 1 maaaaatavgpgagsagvag pggagpcatv svfpgarllt igdangeiqr haeqqalrle 61 vragpdaagialyshedvcv fkcsvsrete csrvgrqsfi itlgcnsvli qfatphdfcs 121 fynilktcrghtlersvfse rteessavqy fqfygvlsqq qnmmqdyvrt gtyqrailqn 181 htdfkdkiaaavgcgsgils ffaaqagark iyaveastma qhaevlvksn nltdrivvip 241 gkveevslpeqvdiiisepm gymlfnerml esylhakkyl kpsgnmfpti gdvhlapftd 301 eqlymeqftkanfryqpsfh gvdlsalrga avdeyfrqpv vdtfdirilm aksvkytvnf 361 leakegdlhrieipfkfhml hsglvhglaf wfdvafigsi mtvwlstapt eplthwyqvr 421 clfqsplfakagdtlsgtcl liankrqsyd isivaqvdqt gskssnlldl knpffrytgt 481 tpspppgshytspsenmwnt gstynlssgv avagmptayd lssviaggss vghnnlipla 541 ntgivnhthsrmgsimstgi vqgssgaqgg ggsssahyav nnqftmggpa ismaspmsip 601 tntmhvgs

TABLE 5 SEQ ID NOS: 4 and 5. Peptides used for in vitro methylationexperiments. R1 peptide SEQ ID NO: 4 GGFGGRGGFG K1 peptide SEQ ID NO: 5GGFGGKGGFG

TABLE 6 SEQ iD NO: 6. Peptide used to generate anti-CARM1 antisera. SEQID NO: 6: CSPMSIPTNTMHYGS

TABLE 7 SEQ D NO: 7. Human PRMT1 (GenBank Accession No. CAA71765). 1mevscgqaes sekpnaedmt skdyyfdsya hfgiheemlk devrtltyrn smfhnrhlfk 61dkvvldvgsg tgilcmfaak agarkvigiv cssisdyavk ivkankldhv vtiikgkvee 121velpvekvdi iisewmgycl fyesmlntvl yardkwlapd glifpdratl yvtaiedrqy 181kdykihwwen vygfdmscik dvaikeplvd vvdpkqlvtn aclikevdiy tvkvedltft 241spfclqvkrn dyvhalvayf nieftrchkr tgfstspesp ythwkqtvfy medyltvktg 301eeifgtigmr pnaknnrdld ftidldfkgq lcelscstdy rmr

TABLE 8 SEQ ID NO: 8. Human PRMT2 (GenBank Accession No. CAA67599) 1matsgdcprs esqgeepaec seagllqegv qpeefvaiad yaatdetqls flrgekilil 61rqttadwwwg eragccgyip anhvgkhvde ydpedtwqde eyfgsygtlk lhlemladqp 121rttkyhsvil qnkesltdkv ildvgcgtgi islfcahyar pravyaveas emaqhtgqlv 161lqngfadiit vyqqkvedvv lpekvdvlvs ewmgtcllfe fmiesilyar dawlkedgvi 241wptmaalhlv pcsadkdyrs kvlfwdnaye fnlsalksla vkeffskpky nhilkpedcl 301sepctilqld mrtvqisdle tlrgelrfdi rkagtlhgft awfsvhfqsl qegqppqvls 361tgpfhptthw kqtlfmmddp vpvhtgdvvt gsvvlqrnpv wrrhmsvals wavtsrgdpt 421sqkvgekvfp iwr

TABLE 9 SEQ ID NO: 9. Human PRMT3 (GenBank Accession No. AAC39837) 1depelsdsgd eaawededda dlphgkqqtp clfcnrlfts aeetfshcks ehqfnidsmv 61hkhglefygy iklinfirlk nptveymnsi ynpvpwekee ylkpvleddl llqfdvedly 121epvsvpfsyp nglsentsvv eklkhmeara lsaeaalara redlqkmkqf aqdfvmhtdv 161rtcssstsvi adlqededgv yfssyghygi heemlkdkir tesyrdfiyq nphifkdkvv 241ldvgcgtgil smfaakagak kvlgvdqsei lyqamdiirl nkledtitli kgkieevhlp 301vekvdviise wmgyfllfes mldsvlyakn kylakggsvy pdictislva vsdvnkhadr 361iafwddvygf kmscmkkavi peavvevldp ktlisepcgi khidchttsi sdlefssdft 421lkitrtsmct aiagyfdiyf eknchnrvvf stgpqstkth wkqtvfllek pfsvkageal 481kgkvtvhknk kdprsltvtl tlnnstqtyg lq

TABLE 10 SEQ ID NO: 10. Yease ODP1 Protein Arginine Methyltransferase.(GenBank Accession No. 6319508) 1 msktavkdsa tektklsese qhyfnsydhygiheemlqdt vrtlsyrnai iqnkdlfkdk 61 ivldvgcgtg ilsmfaakhg akhvigvdmssiiemakelv elngfsdkit llrgkledvh 121 lpfpkvdiii sewmgyflly esmmdtvlyardhylveggl ifpdkcsihl agledsqykd 181 eklnywqdvy gfdyspfvpl vlhepivdtvernnvnttsd kliefdlntv kisdlafksn 241 fkltakrqdm ingivtwfdi vfpapkgkrpvefstgphap ythwkqtify fpddldaetg 301 dtiegelvcs pneknnrdln ikisykfesngidgnsrsrk negsylmh

10 1 3124 DNA Mus musculuc 1 agggggcctg gagccggacc taagatggca gcggcggcagcgacggcggt ggggccgggt 60 gcggggagcg ctggggtggc gggcccgggc ggcgcggggccctgcgctac agtgtctgtg 120 ttcccgggcg cccgcctcct cactatcggc gacgcgaacggcgagatcca gcggcacgcg 180 gagcagcagg cgctgcgcct tgaggtgcgc gccggaccagacgcggcggg catcgccctc 240 tacagccatg aagatgtgtg tgttttcaag tgctcggtgtcccgagagac agagtgcagt 300 cgtgtgggca gacagtcctt catcatcacc ctgggctgcaacagcgtcct catccagttt 360 gccacacccc acgatttctg ttctttctac aacatcctgaaaacctgtcg gggccacaca 420 ctggagcgct ctgtgttcag tgagcggaca gaggaatcctcagctgtgca gtacttccag 480 ttctatggct acctatccca gcagcagaac atgatgcaggactatgtgcg gacaggcacc 540 taccagcgtg cgatcctgca gaaccacacg gacttcaaggacaagatcgt tctagatgtg 600 ggctgtggct ctgggatcct gtcatttttt gctgctcaagcaggagccag gaaaatttat 660 gcagtggaag ccagcaccat ggctcagcat gcagaggtcctggtgaagag taacaatctg 720 acagaccgca tcgtggtcat ccctggcaaa gtagaggaggtctcattgcc tgagcaagtg 780 gacattatca tctcagagcc catgggctac atgctcttcaatgaacgaat gctcgagagc 840 tacctccatg ccaaaaagta cctgaagcct agtggaaacatgttccccac cattggtgat 900 gtccacctcg cacccttcac tgatgaacag ctctacatggagcagttcac caaagccaac 960 ttccggtacc agccatcctt ccatggagtg gacctgtcggccctcagagg tgccgctgtg 1020 gatgagtact tccggcaacc tgtggtggac acatttgacatccggatcct gatggccaaa 1080 tctgtcaagt acacagtgaa cttcttagaa gccaaagaaggcgatttgca caggatagaa 1140 atcccattca aattccacat gctgcattca gggctagtccatggcttggc cttctggttc 1200 gatgttgctt tcattggctc cataatgacc gtgtggctatccacagcccc aacagagccc 1260 ctgacccact ggtaccaggt ccggtgcctc ttccagtcaccgttgtttgc caaggccggg 1320 gacacgctct cagggacatg tctgcttatt gccaacaaaagacagagcta tgacatcagt 1380 attgtggcac aggtggacca gacaggctcc aagtccagtaacctgctgga tctaaagaac 1440 cccttcttca ggtacacagg tacaacccca tcacccccacctggctcaca ctacacgtct 1500 ccctcggaga atatgtggaa cacaggaagc acctataatctcagcagcgg ggtggctgtg 1560 gctggaatgc ctactgccta cgacctgagc agtgttattgccggcggctc cagtgtgggt 1620 cacaacaacc tgattccctt agctaacaca gggattgtcaatcacaccca ctcccggatg 1680 ggctccataa tgagcacggg cattgtccaa ggctcctcaggtgcccaggg aggcggcggt 1740 agctccagtg cccactatgc agtcaacaac cagttcaccatgggtggccc tgccatctct 1800 atggcctcgc ccatgtccat cccgaccaac accatgcactatgggagtta ggtgcctcca 1860 gccgcgacag cactgcgcac tgacagcacc aggaaaccaaatcaagtcca ggcccggcac 1920 agccagtggc tgttccccct tgttctggag aagttgttgaacacccggtc acagcctcct 1980 tgctatggga acttggacaa ttttgtacac gatgtcgccgctgccctcaa gtacccccag 2040 cccaaccttt ggtcccgagc gcgtgttgct gccatactttacatgagatc ctgttggggc 2100 agccctcatc ctgttctgta ctctccactc tgacctggctttgacatctg ctggaagagg 2160 caagtcctcc cccaaccccc acagctgcac ctgaccaggcaggaggaggc cagcagctgc 2220 caccacagac ctggcagcac ccaccccaca acccgtccttgcacctcccc tcacctgggg 2280 tggcagcaca gccagctgga cctctccttc aactaccaggccacatggtc accatgggcg 2340 tgacatgctg ctttttttaa ttttattttt ttacgaaaagaaccagtgtc aacccacaga 2400 ccctctgaga aacccggctg gcgcgccaag ccagcagcccctgttcctag gcccagaggt 2460 tctaggtgag gggtggccct gtcaagcctt cagagtgggcacagcccctc ccaccaaagg 2520 gttcacctca aacttgaatg tacaaaccac ccagctgtccaaaggcctag tccctacttt 2580 ctgctactgt cctgtcctga gccctgaagg cccccctccatcaaaagctt gaacaggcag 2640 cccagagtgt gtcaccctgg gctactgggg cagacaagaaacctcaaaga tctgtcacac 2700 acacacaagg aaggcgtcct ctcctgatag ctgacataggcctgtgtgtt gcgttcacat 2760 tcatgttcta cttaatcctc tcaagacagc aaccctgggaaggagcctcg cagggacctc 2820 cccagacaag aagaaaagca aacaaggaag ggtgattaataagcacaggc agtttcccct 2880 attcccttac cctagagtcc ccacctgaat ggccacagcctgccacagga accccttggc 2940 aaaggctgga gctgctctgt gccaccctcc tgacctgtcagggaatcaca gggccctcag 3000 gcagctggga accaggctct ctcctgtcca tcagtaatactccttgctcg gatggccctc 3060 ccccaccttt atataaattc tctggatcac ctttgcatagaaaataaaag tgtttgcttt 3120 gtaa 3124 2 608 PRT Artificial SequenceDeduced amino acid sequence of CARM1 2 Met Ala Ala Ala Ala Ala Thr AlaVal Gly Pro Gly Ala Gly Ser Ala 1 5 10 15 Gly Val Ala Gly Pro Gly GlyAla Gly Pro Cys Ala Thr Val Ser Val 20 25 30 Phe Pro Gly Ala Arg Leu LeuThr Ile Gly Asp Ala Asn Gly Glu Ile 35 40 45 Gln Arg His Ala Glu Gln GlnAla Leu Arg Leu Glu Val Arg Ala Gly 50 55 60 Pro Asp Ala Ala Gly Ile AlaLeu Tyr Ser His Glu Asp Val Cys Val 65 70 75 80 Phe Lys Cys Ser Val SerArg Glu Thr Glu Cys Ser Arg Val Gly Arg 85 90 95 Gln Ser Phe Ile Ile ThrLeu Gly Cys Asn Ser Val Leu Ile Gln Phe 100 105 110 Ala Thr Pro His AspPhe Cys Ser Phe Tyr Asn Ile Leu Lys Thr Cys 115 120 125 Arg Gly His ThrLeu Glu Arg Ser Val Phe Ser Glu Arg Thr Glu Glu 130 135 140 Ser Ser AlaVal Gln Tyr Phe Gln Phe Tyr Gly Tyr Leu Ser Gln Gln 145 150 155 160 GlnAsn Met Met Gln Asp Tyr Val Arg Thr Gly Thr Tyr Gln Arg Ala 165 170 175Ile Leu Gln Asn His Thr Asp Phe Lys Asp Lys Ile Val Leu Asp Val 180 185190 Gly Cys Gly Ser Gly Ile Leu Ser Phe Phe Ala Ala Gln Ala Gly Ala 195200 205 Arg Lys Ile Tyr Ala Val Glu Ala Ser Thr Met Ala Gln His Ala Glu210 215 220 Val Leu Val Lys Ser Asn Asn Leu Thr Asp Arg Ile Val Val IlePro 225 230 235 240 Gly Lys Val Glu Glu Val Ser Leu Pro Glu Gln Val AspIle Ile Ile 245 250 255 Ser Glu Pro Met Gly Tyr Met Leu Phe Asn Glu ArgMet Leu Glu Ser 260 265 270 Tyr Leu His Ala Lys Lys Tyr Leu Lys Pro SerGly Asn Met Phe Pro 275 280 285 Thr Ile Gly Asp Val His Leu Ala Pro PheThr Asp Glu Gln Leu Tyr 290 295 300 Met Glu Gln Phe Thr Lys Ala Asn PheArg Tyr Gln Pro Ser Phe His 305 310 315 320 Gly Val Asp Leu Ser Ala LeuArg Gly Ala Ala Val Asp Glu Tyr Phe 325 330 335 Arg Gln Pro Val Val AspThr Phe Asp Ile Arg Ile Leu Met Ala Lys 340 345 350 Ser Val Lys Tyr ThrVal Asn Phe Leu Glu Ala Lys Glu Gly Asp Leu 355 360 365 His Arg Ile GluIle Pro Phe Lys Phe His Met Leu His Ser Gly Leu 370 375 380 Val His GlyLeu Ala Phe Trp Phe Asp Val Ala Phe Ile Gly Ser Ile 385 390 395 400 MetThr Val Trp Leu Ser Thr Ala Pro Thr Glu Pro Leu Thr His Trp 405 410 415Tyr Gln Val Arg Cys Leu Phe Gln Ser Pro Leu Phe Ala Lys Ala Gly 420 425430 Asp Thr Leu Ser Gly Thr Cys Leu Leu Ile Ala Asn Lys Arg Gln Ser 435440 445 Tyr Asp Ile Ser Ile Val Ala Gln Val Asp Gln Thr Gly Ser Lys Ser450 455 460 Ser Asn Leu Leu Asp Leu Lys Asn Pro Phe Phe Arg Tyr Thr GlyThr 465 470 475 480 Thr Pro Ser Pro Pro Pro Gly Ser His Tyr Thr Ser ProSer Glu Asn 485 490 495 Met Trp Asn Thr Gly Ser Thr Tyr Asn Leu Ser SerGly Val Ala Val 500 505 510 Ala Gly Met Pro Thr Ala Tyr Asp Leu Ser SerVal Ile Ala Gly Gly 515 520 525 Ser Ser Val Gly His Asn Asn Leu Ile ProLeu Ala Asn Thr Gly Ile 530 535 540 Val Asn His Thr His Ser Arg Met GlySer Ile Met Ser Thr Gly Ile 545 550 555 560 Val Gln Gly Ser Ser Gly AlaGln Gly Gly Gly Gly Ser Ser Ser Ala 565 570 575 His Tyr Ala Val Asn AsnGln Phe Thr Met Gly Gly Pro Ala Ile Ser 580 585 590 Met Ala Ser Pro MetSer Ile Pro Thr Asn Thr Met His Tyr Gly Ser 595 600 605 3 608 PRTArtificial Sequence CARM1 VLD TO AAA Variant 3 Met Ala Ala Ala Ala AlaThr Ala Val Gly Pro Gly Ala Gly Ser Ala 1 5 10 15 Gly Val Ala Gly ProGly Gly Ala Gly Pro Cys Ala Thr Val Ser Val 20 25 30 Phe Pro Gly Ala ArgLeu Leu Thr Ile Gly Asp Ala Asn Gly Glu Ile 35 40 45 Gln Arg His Ala GluGln Gln Ala Leu Arg Leu Glu Val Arg Ala Gly 50 55 60 Pro Asp Ala Ala GlyIle Ala Leu Tyr Ser His Glu Asp Val Cys Val 65 70 75 80 Phe Lys Cys SerVal Ser Arg Glu Thr Glu Cys Ser Arg Val Gly Arg 85 90 95 Gln Ser Phe IleIle Thr Leu Gly Cys Asn Ser Val Leu Ile Gln Phe 100 105 110 Ala Thr ProHis Asp Phe Cys Ser Phe Tyr Asn Ile Leu Lys Thr Cys 115 120 125 Arg GlyHis Thr Leu Glu Arg Ser Val Phe Ser Glu Arg Thr Glu Glu 130 135 140 SerSer Ala Val Gln Tyr Phe Gln Phe Tyr Gly Tyr Leu Ser Gln Gln 145 150 155160 Gln Asn Met Met Gln Asp Tyr Val Arg Thr Gly Thr Tyr Gln Arg Ala 165170 175 Ile Leu Gln Asn His Thr Asp Phe Lys Asp Lys Ile Ala Ala Ala Val180 185 190 Gly Cys Gly Ser Gly Ile Leu Ser Phe Phe Ala Ala Gln Ala GlyAla 195 200 205 Arg Lys Ile Tyr Ala Val Glu Ala Ser Thr Met Ala Gln HisAla Glu 210 215 220 Val Leu Val Lys Ser Asn Asn Leu Thr Asp Arg Ile ValVal Ile Pro 225 230 235 240 Gly Lys Val Glu Glu Val Ser Leu Pro Glu GlnVal Asp Ile Ile Ile 245 250 255 Ser Glu Pro Met Gly Tyr Met Leu Phe AsnGlu Arg Met Leu Glu Ser 260 265 270 Tyr Leu His Ala Lys Lys Tyr Leu LysPro Ser Gly Asn Met Phe Pro 275 280 285 Thr Ile Gly Asp Val His Leu AlaPro Phe Thr Asp Glu Gln Leu Tyr 290 295 300 Met Glu Gln Phe Thr Lys AlaAsn Phe Arg Tyr Gln Pro Ser Phe His 305 310 315 320 Gly Val Asp Leu SerAla Leu Arg Gly Ala Ala Val Asp Glu Tyr Phe 325 330 335 Arg Gln Pro ValVal Asp Thr Phe Asp Ile Arg Ile Leu Met Ala Lys 340 345 350 Ser Val LysTyr Thr Val Asn Phe Leu Glu Ala Lys Glu Gly Asp Leu 355 360 365 His ArgIle Glu Ile Pro Phe Lys Phe His Met Leu His Ser Gly Leu 370 375 380 ValHis Gly Leu Ala Phe Trp Phe Asp Val Ala Phe Ile Gly Ser Ile 385 390 395400 Met Thr Val Trp Leu Ser Thr Ala Pro Thr Glu Pro Leu Thr His Trp 405410 415 Tyr Gln Val Arg Cys Leu Phe Gln Ser Pro Leu Phe Ala Lys Ala Gly420 425 430 Asp Thr Leu Ser Gly Thr Cys Leu Leu Ile Ala Asn Lys Arg GlnSer 435 440 445 Tyr Asp Ile Ser Ile Val Ala Gln Val Asp Gln Thr Gly SerLys Ser 450 455 460 Ser Asn Leu Leu Asp Leu Lys Asn Pro Phe Phe Arg TyrThr Gly Thr 465 470 475 480 Thr Pro Ser Pro Pro Pro Gly Ser His Tyr ThrSer Pro Ser Glu Asn 485 490 495 Met Trp Asn Thr Gly Ser Thr Tyr Asn LeuSer Ser Gly Val Ala Val 500 505 510 Ala Gly Met Pro Thr Ala Tyr Asp LeuSer Ser Val Ile Ala Gly Gly 515 520 525 Ser Ser Val Gly His Asn Asn LeuIle Pro Leu Ala Asn Thr Gly Ile 530 535 540 Val Asn His Thr His Ser ArgMet Gly Ser Ile Met Ser Thr Gly Ile 545 550 555 560 Val Gln Gly Ser SerGly Ala Gln Gly Gly Gly Gly Ser Ser Ser Ala 565 570 575 His Tyr Ala ValAsn Asn Gln Phe Thr Met Gly Gly Pro Ala Ile Ser 580 585 590 Met Ala SerPro Met Ser Ile Pro Thr Asn Thr Met His Tyr Gly Ser 595 600 605 4 10 PRTArtifical Sequence Peptide used for in vitro methylation experiments 4Gly Gly Phe Gly Gly Arg Gly Gly Phe Gly 1 5 10 5 10 PRT ArtificialSequence Peptide used for in vitro methylation experiments 5 Gly Gly PheGly Gly Lys Gly Gly Phe Gly 1 5 10 6 15 PRT Artificial Sequence Peptideused to generate anti-CARM1 antisera 6 Cys Ser Pro Met Ser Ile Pro ThrAsn Thr Met His Tyr Gly Ser 1 5 10 15 7 343 PRT Artifical Sequence HumanPRMT1 7 Met Glu Val Ser Cys Gly Gln Ala Glu Ser Ser Glu Lys Pro Asn Ala1 5 10 15 Glu Asp Met Thr Ser Lys Asp Tyr Tyr Phe Asp Ser Tyr Ala HisPhe 20 25 30 Gly Ile His Glu Glu Met Leu Lys Asp Glu Val Arg Thr Leu ThrTyr 35 40 45 Arg Asn Ser Met Phe His Asn Arg His Leu Phe Lys Asp Lys ValVal 50 55 60 Leu Asp Val Gly Ser Gly Thr Gly Ile Leu Cys Met Phe Ala AlaLys 65 70 75 80 Ala Gly Ala Arg Lys Val Ile Gly Ile Val Cys Ser Ser IleSer Asp 85 90 95 Tyr Ala Val Lys Ile Val Lys Ala Asn Lys Leu Asp His ValVal Thr 100 105 110 Ile Ile Lys Gly Lys Val Glu Glu Val Glu Leu Pro ValGlu Lys Val 115 120 125 Asp Ile Ile Ile Ser Glu Trp Met Gly Tyr Cys LeuPhe Tyr Glu Ser 130 135 140 Met Leu Asn Thr Val Leu Tyr Ala Arg Asp LysTrp Leu Ala Pro Asp 145 150 155 160 Gly Leu Ile Phe Pro Asp Arg Ala ThrLeu Tyr Val Thr Ala Ile Glu 165 170 175 Asp Arg Gln Tyr Lys Asp Tyr LysIle His Trp Trp Glu Asn Val Tyr 180 185 190 Gly Phe Asp Met Ser Cys IleLys Asp Val Ala Ile Lys Glu Pro Leu 195 200 205 Val Asp Val Val Asp ProLys Gln Leu Val Thr Asn Ala Cys Leu Ile 210 215 220 Lys Glu Val Asp IleTyr Thr Val Lys Val Glu Asp Leu Thr Phe Thr 225 230 235 240 Ser Pro PheCys Leu Gln Val Lys Arg Asn Asp Tyr Val His Ala Leu 245 250 255 Val AlaTyr Phe Asn Ile Glu Phe Thr Arg Cys His Lys Arg Thr Gly 260 265 270 PheSer Thr Ser Pro Glu Ser Pro Tyr Thr His Trp Lys Gln Thr Val 275 280 285Phe Tyr Met Glu Asp Tyr Leu Thr Val Lys Thr Gly Glu Glu Ile Phe 290 295300 Gly Thr Ile Gly Met Arg Pro Asn Ala Lys Asn Asn Arg Asp Leu Asp 305310 315 320 Phe Thr Ile Asp Leu Asp Phe Lys Gly Gln Leu Cys Glu Leu SerCys 325 330 335 Ser Thr Asp Tyr Arg Met Arg 340 8 433 PRT ArtificialSequence Human PRMT2 8 Met Ala Thr Ser Gly Asp Cys Pro Arg Ser Glu SerGln Gly Glu Glu 1 5 10 15 Pro Ala Glu Cys Ser Glu Ala Gly Leu Leu GlnGlu Gly Val Gln Pro 20 25 30 Glu Glu Phe Val Ala Ile Ala Asp Tyr Ala AlaThr Asp Glu Thr Gln 35 40 45 Leu Ser Phe Leu Arg Gly Glu Lys Ile Leu IleLeu Arg Gln Thr Thr 50 55 60 Ala Asp Trp Trp Trp Gly Glu Arg Ala Gly CysCys Gly Tyr Ile Pro 65 70 75 80 Ala Asn His Val Gly Lys His Val Asp GluTyr Asp Pro Glu Asp Thr 85 90 95 Trp Gln Asp Glu Glu Tyr Phe Gly Ser TyrGly Thr Leu Lys Leu His 100 105 110 Leu Glu Met Leu Ala Asp Gln Pro ArgThr Thr Lys Tyr His Ser Val 115 120 125 Ile Leu Gln Asn Lys Glu Ser LeuThr Asp Lys Val Ile Leu Asp Val 130 135 140 Gly Cys Gly Thr Gly Ile IleSer Leu Phe Cys Ala His Tyr Ala Arg 145 150 155 160 Pro Arg Ala Val TyrAla Val Glu Ala Ser Glu Met Ala Gln His Thr 165 170 175 Gly Gln Leu ValLeu Gln Asn Gly Phe Ala Asp Ile Ile Thr Val Tyr 180 185 190 Gln Gln LysVal Glu Asp Val Val Leu Pro Glu Lys Val Asp Val Leu 195 200 205 Val SerGlu Trp Met Gly Thr Cys Leu Leu Phe Glu Phe Met Ile Glu 210 215 220 SerIle Leu Tyr Ala Arg Asp Ala Trp Leu Lys Glu Asp Gly Val Ile 225 230 235240 Trp Pro Thr Met Ala Ala Leu His Leu Val Pro Cys Ser Ala Asp Lys 245250 255 Asp Tyr Arg Ser Lys Val Leu Phe Trp Asp Asn Ala Tyr Glu Phe Asn260 265 270 Leu Ser Ala Leu Lys Ser Leu Ala Val Lys Glu Phe Phe Ser LysPro 275 280 285 Lys Tyr Asn His Ile Leu Lys Pro Glu Asp Cys Leu Ser GluPro Cys 290 295 300 Thr Ile Leu Gln Leu Asp Met Arg Thr Val Gln Ile SerAsp Leu Glu 305 310 315 320 Thr Leu Arg Gly Glu Leu Arg Phe Asp Ile ArgLys Ala Gly Thr Leu 325 330 335 His Gly Phe Thr Ala Trp Phe Ser Val HisPhe Gln Ser Leu Gln Glu 340 345 350 Gly Gln Pro Pro Gln Val Leu Ser ThrGly Pro Phe His Pro Thr Thr 355 360 365 His Trp Lys Gln Thr Leu Phe MetMet Asp Asp Pro Val Pro Val His 370 375 380 Thr Gly Asp Val Val Thr GlySer Val Val Leu Gln Arg Asn Pro Val 385 390 395 400 Trp Arg Arg His MetSer Val Ala Leu Ser Trp Ala Val Thr Ser Arg 405 410 415 Gln Asp Pro ThrSer Gln Lys Val Gly Glu Lys Val Phe Pro Ile Trp 420 425 430 Arg 9 512PRT Artificial Sequence Human PRMT3 9 Asp Glu Pro Glu Leu Ser Asp SerGly Asp Glu Ala Ala Trp Glu Asp 1 5 10 15 Glu Asp Asp Ala Asp Leu ProHis Gly Lys Gln Gln Thr Pro Cys Leu 20 25 30 Phe Cys Asn Arg Leu Phe ThrSer Ala Glu Glu Thr Phe Ser His Cys 35 40 45 Lys Ser Glu His Gln Phe AsnIle Asp Ser Met Val His Lys His Gly 50 55 60 Leu Glu Phe Tyr Gly Tyr IleLys Leu Ile Asn Phe Ile Arg Leu Lys 65 70 75 80 Asn Pro Thr Val Glu TyrMet Asn Ser Ile Tyr Asn Pro Val Pro Trp 85 90 95 Glu Lys Glu Glu Tyr LeuLys Pro Val Leu Glu Asp Asp Leu Leu Leu 100 105 110 Gln Phe Asp Val GluAsp Leu Tyr Glu Pro Val Ser Val Pro Phe Ser 115 120 125 Tyr Pro Asn GlyLeu Ser Glu Asn Thr Ser Val Val Glu Lys Leu Lys 130 135 140 His Met GluAla Arg Ala Leu Ser Ala Glu Ala Ala Leu Ala Arg Ala 145 150 155 160 ArgGlu Asp Leu Gln Lys Met Lys Gln Phe Ala Gln Asp Phe Val Met 165 170 175His Thr Asp Val Arg Thr Cys Ser Ser Ser Thr Ser Val Ile Ala Asp 180 185190 Leu Gln Glu Asp Glu Asp Gly Val Tyr Phe Ser Ser Tyr Gly His Tyr 195200 205 Gly Ile His Glu Glu Met Leu Lys Asp Lys Ile Arg Thr Glu Ser Tyr210 215 220 Arg Asp Phe Ile Tyr Gln Asn Pro His Ile Phe Lys Asp Lys ValVal 225 230 235 240 Leu Asp Val Gly Cys Gly Thr Gly Ile Leu Ser Met PheAla Ala Lys 245 250 255 Ala Gly Ala Lys Lys Val Leu Gly Val Asp Gln SerGlu Ile Leu Tyr 260 265 270 Gln Ala Met Asp Ile Ile Arg Leu Asn Lys LeuGlu Asp Thr Ile Thr 275 280 285 Leu Ile Lys Gly Lys Ile Glu Glu Val HisLeu Pro Val Glu Lys Val 290 295 300 Asp Val Ile Ile Ser Glu Trp Met GlyTyr Phe Leu Leu Phe Glu Ser 305 310 315 320 Met Leu Asp Ser Val Leu TyrAla Lys Asn Lys Tyr Leu Ala Lys Gly 325 330 335 Gly Ser Val Tyr Pro AspIle Cys Thr Ile Ser Leu Val Ala Val Ser 340 345 350 Asp Val Asn Lys HisAla Asp Arg Ile Ala Phe Trp Asp Asp Val Tyr 355 360 365 Gly Phe Lys MetSer Cys Met Lys Lys Ala Val Ile Pro Glu Ala Val 370 375 380 Val Glu ValLeu Asp Pro Lys Thr Leu Ile Ser Glu Pro Cys Gly Ile 385 390 395 400 LysHis Ile Asp Cys His Thr Thr Ser Ile Ser Asp Leu Glu Phe Ser 405 410 415Ser Asp Phe Thr Leu Lys Ile Thr Arg Thr Ser Met Cys Thr Ala Ile 420 425430 Ala Gly Tyr Phe Asp Ile Tyr Phe Glu Lys Asn Cys His Asn Arg Val 435440 445 Val Phe Ser Thr Gly Pro Gln Ser Thr Lys Thr His Trp Lys Gln Thr450 455 460 Val Phe Leu Leu Glu Lys Pro Phe Ser Val Lys Ala Gly Glu AlaLeu 465 470 475 480 Lys Gly Lys Val Thr Val His Lys Asn Lys Lys Asp ProArg Ser Leu 485 490 495 Thr Val Thr Leu Thr Leu Asn Asn Ser Thr Gln ThrTyr Gly Leu Gln 500 505 510 10 348 PRT Artificial Sequence Yeast ODP1Protein Arginine Methyltransferase 10 Met Ser Lys Thr Ala Val Lys AspSer Ala Thr Glu Lys Thr Lys Leu 1 5 10 15 Ser Glu Ser Glu Gln His TyrPhe Asn Ser Tyr Asp His Tyr Gly Ile 20 25 30 His Glu Glu Met Leu Gln AspThr Val Arg Thr Leu Ser Tyr Arg Asn 35 40 45 Ala Ile Ile Gln Asn Lys AspLeu Phe Lys Asp Lys Ile Val Leu Asp 50 55 60 Val Gly Cys Gly Thr Gly IleLeu Ser Met Phe Ala Ala Lys His Gly 65 70 75 80 Ala Lys His Val Ile GlyVal Asp Met Ser Ser Ile Ile Glu Met Ala 85 90 95 Lys Glu Leu Val Glu LeuAsn Gly Phe Ser Asp Lys Ile Thr Leu Leu 100 105 110 Arg Gly Lys Leu GluAsp Val His Leu Pro Phe Pro Lys Val Asp Ile 115 120 125 Ile Ile Ser GluTrp Met Gly Tyr Phe Leu Leu Tyr Glu Ser Met Met 130 135 140 Asp Thr ValLeu Tyr Ala Arg Asp His Tyr Leu Val Glu Gly Gly Leu 145 150 155 160 IlePhe Pro Asp Lys Cys Ser Ile His Leu Ala Gly Leu Glu Asp Ser 165 170 175Gln Tyr Lys Asp Glu Lys Leu Asn Tyr Trp Gln Asp Val Tyr Gly Phe 180 185190 Asp Tyr Ser Pro Phe Val Pro Leu Val Leu His Glu Pro Ile Val Asp 195200 205 Thr Val Glu Arg Asn Asn Val Asn Thr Thr Ser Asp Lys Leu Ile Glu210 215 220 Phe Asp Leu Asn Thr Val Lys Ile Ser Asp Leu Ala Phe Lys SerAsn 225 230 235 240 Phe Lys Leu Thr Ala Lys Arg Gln Asp Met Ile Asn GlyIle Val Thr 245 250 255 Trp Phe Asp Ile Val Phe Pro Ala Pro Lys Gly LysArg Pro Val Glu 260 265 270 Phe Ser Thr Gly Pro His Ala Pro Tyr Thr HisTrp Lys Gln Thr Ile 275 280 285 Phe Tyr Phe Pro Asp Asp Leu Asp Ala GluThr Gly Asp Thr Ile Glu 290 295 300 Gly Glu Leu Val Cys Ser Pro Asn GluLys Asn Asn Arg Asp Leu Asn 305 310 315 320 Ile Lys Ile Ser Tyr Lys PheGlu Ser Asn Gly Ile Asp Gly Asn Ser 325 330 335 Arg Ser Arg Lys Asn GluGly Ser Tyr Leu Met His 340 345

What is claimed is:
 1. An isolated nucleic acid molecule comprising asequence that has at least about 90% sequence identity to SEQ ID NO:1 ora complementary sequence thereof, wherein the nucleic acid encodes apolypeptide that binds a C terminus of GRIP1.
 2. A recombinant vectorcomprising the nucleic acid molecule of claim
 1. 3. A geneticallyengineered cell comprising the recombinant vector of claim
 2. 4. Theisolated nucleic acid molecule of claim 1, wherein said nucleic acidmolecule encodes a polypeptide comprising SEQ ID NO:2.
 5. A recombinantvector comprising the nucleic acid molecule of claim
 4. 6. A geneticallyengineered cell comprising the recombinant vector of claim
 5. 7. Theisolated nucleic acid molecule of claim 1, wherein said nucleic acidmolecule comprises a sequence that has at least about 95% sequenceidentity to SEQ ID NO:1.
 8. A recombinant vector comprising the nucleicacid molecule of claim
 7. 9. A genetically engineered cell comprisingthe recombinant vector of claim
 8. 10. The isolated nucleic acidmolecule of claim 1, wherein said nucleic acid molecule comprises SEQ IDNO:1.
 11. A recombinant vector comprising the nucleic acid molecule ofclaim
 10. 12. A genetically engineered cell comprising the recombinantvector of claim
 11. 13. The isolated nucleic acid molecule of claim 1,wherein the polypeptide comprises methyltransferase activity.
 14. Theisolated nucleic acid molecule of claim 1, wherein the polypeptidecomprises coactivator activity.